Dosage regime and method for treating pulmonary arterial hypertension

ABSTRACT

There is provided a daily dosage regimen for treating pulmonary arterial hypertension. The regimen takes the form of two discrete dosage forms. Each dosage form includes an amount of about 600 mg to about 800 mg of (S)-ethyl 8-(2-amino-6-((R)-1-(5-chloro-[1,1′-biphenyl]-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylate. There is also another method for providing a daily dosage regimen. There is also provided several methods for treating pulmonary arterial hypertension. There is provided a method for reducing the level of serotonin biosynthesis by at least 50%. There is also provided a method for achieving an AUC 0-tau  of ≥2530 ng·hr/mL of (S)-8-(2-amino-6-((R)-1-(5-chloro-[1,1′-biphenyl]-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylic acid. There is provided a method of achieving a &gt;50% reduction in urinary 5-HIAA.

CROSS-REFERENCE TO A RELATED APPLICATION

The present application is a continuation of U.S. patent application Ser. No. 16/777,458, filed on Jan. 30, 2020, which claims priority to U.S. Provisional Application No. 62/798827, filed Jan. 30, 2019. The entire contents of the foregoing applications are incorporated herein by reference in entirety.

BACKGROUND OF THE DISCLOSURE 1. Field of the Disclosure

The present disclosure relates to a daily dosage regimen for treating pulmonary arterial hypertension. The present disclosure also relates to methods for treating pulmonary arterial hypertension. The present disclosure further relates to a method for reducing the level of serotonin biosynthesis within a period of time. The present disclosure still further relates to a method for achieving an enhanced level of AUC_(0-tau) within a period of time. The present disclosure further relates to a method for achieving a reduction in urinary 5-HIAA within a period of time.

2. Description of the Prior Art

Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter that modulates central and peripheral functions by acting on neurons, smooth muscle, and other cell types. 5-HT is involved in the control and modulation of multiple physiological and psychological processes. In the central nervous system (CNS), 5-HT regulates mood, appetite, and other behavioral functions. In the GI system, 5-HT has a general prokinetic role and is an important mediator of sensation (e.g., nausea and satiety) between the GI tract and the brain. Dysregulation of the peripheral 5-HT signaling system has been reported to be involved in the etiology of several conditions (see for example: Mawe, G. M. & Hoffman, J., Serotonin Signaling in the Gut-functions, Dysfunctions and Therapeutic Targets. Nature Reviews. Gastroenterology & Hepatology 10, 473-486 (2013); Gershon, M. D. 5-hydroxytryptamine (serotonin) in the Gastrointestinal Tract. Current Opinion in Endocrinology, Diabetes, and Obesity 20, 14-21 (2013); Lesurtel, M., Soil, C, Graf, R. & Ciavien, P.-A. Role of Serotonin in the Hepato-gastrointestinal Tract: An Old Molecule for New Perspectives. Cellular and Molecular Life Sciences: CMLS 65, 940-52 (2008)). These include osteoporosis (e.g. Kode, A, et al., FOXO1 Orchestrates the Bone-suppressing Function of Gut-derived Serotonin, The Journal of Clinical Investigation 122, 3490-503 (2012); Yadav, V, K. et al., Pharmacological Inhibition of Gut-derived Serotonin Synthesis is a Potential Bone Anabolic Treatment for Osteoporosis. Nature Medicine 16, 308-12 (2010); Yadav, V. K, et al., Lrp5 Controls Bone Formation by Inhibiting Serotonin Synthesis in the Duodenum, Cell 135, 825-37 (2008), cancer (e.g. Liang, C, et al., Serotonin Promotes the Proliferation of Serum-deprived Hepatocellular Carcinoma Cells Via Upregulation of FOX03a. Molecular Cancer 12, 14 (2013); Soli, C. et al., Serotonin Promotes Tumor Growth in Human Hepatocellular Cancer. Hepatology 51, 1244-1254 (2010); Pai, V. P et al., Altered Serotonin Physiology in Human Breast Cancers Favors Paradoxical Growth and Cell Survival. Breast Cancer Research: BCR 11, R81 (2009); Engelman, K., Lovenberg, W. & Sjoerdsma, A. Inhibition of Serotonin Synthesis by Para-chlorophenylalanine in Patients with The Carcinoid Syndrome. The New England Journal of Medicine 277, 1103-8 (1967)), cardiovascular (e.g. Robiolio, P. A, et al., Carcinoid Heart Disease: Correlation of High Serotonin Levels with Valvular Abnormalities Detected by Cardiac Catheterization and Echocardiography. Circulation 92, 790-795 (1995), diabetes (e.g. Sumara, G., Sumara, O., Kim, J. K. & Karsenty, G. Gut-derived Serotonin is a Multifunctional Determinant to Fasting Adaptation. Cell Metabolism 16, 588-600 (2012), atherosclerosis (e.g. Ban, Y. et al., Impact of Increased Plasma Serotonin Levels and Carotid Atherosclerosis on Vascular Dementia. Atherosclerosis 195, 153-9 (2007), as well as gastrointestinal (e.g. Manocha, M. & Khan, W. I. Serotonin and GI Disorders: An Update on Clinical and Experimental Studies. Clinical and Translational Gastroenterology 3, el 3 (2012); Ghia, J.-E. et al., Serotonin has a Key Role in Pathogenesis of Experimental Colitis. Gastroenterology 137, 1649-60 (2009); Sikander, A., Rana, S. V. & Prasad, K. K., Role of Serotonin in Gastrointestinal Motility and Irritable Bowel Syndrome. Clinica Chimica Acta; International Journal of Clinical Chemistry 403, 47-55 (2009); Spiller, R, Recent Advances in Understanding the Role of Serotonin in Gastrointestinal Motility in Functional Bowel Disorders: Alterations In 5-HT Signaling and Metabolism in Human Disease. Neurogastroenterology and Motility: The Official Journal of The European Gastrointestinal Motility Society 19 Suppl 2, 25-31 (2007); Costedio, M. M., Hyman, N. & Mawe, G, M, Serotonin and its Role in Colonic Function and In Gastrointestinal Disorders. Diseases of the Colon and Rectum 50, 376-88 (2007); Gershon, M. D. & Tack, J., The Serotonin Signaling System: From Basic Understanding to Drug Development for Functional GI Disorders. Gastroenterology 132, 397-14 (2007); Mawe, G. M., Coates, M. D. & Moses, P. L. Review Article: Intestinal Serotonin Signaling In Irritable Bowel Syndrome. Alimentary Pharmacology & Therapeutics 23, 1067-76 (2006); Crowell, M. D. Role of Serotonin in the Pathophysiology of The Irritable Bowel Syndrome. British Journal of Pharmacology 141, 1285-93 (2004)), pulmonary (e.g. Lau, W. K. W. et al., The Role of Circulating Serotonin in the Development of Chronic Obstructive Pulmonary Disease. PloS One 7, e31617 (2012); Egermayer, P., Town, G. I. & Peacock, A. J. Role of Serotonin in the Pathogenesis of Acute and Chronic Pulmonary Hypertension. Thorax 54, 161-168 (1999), inflammatory (e.g. Margolis, K. G. et al., Pharmacological Reduction of Mucosal but Not Neuronal Serotonin Opposes Inflammation in Mouse Intestine. Gut doi: 10.1 136/gutjnl-2013-304901 (2013); Duerschmied, D. et al., Platelet Serotonin Promotes the Recruitment of Neutrophils to Sites of Acute Inflammation in Mice. Blood 121, 1008-15 (2013); Li, N. et al., Serotonin Activates Dendritic Cell Function in the Context of Gut Inflammation. The American Journal of Pathology 178, 662-71 (2011), or liver diseases or disorders (e.g. Ebrahimkhani, M. R. et al., Stimulating Healthy Tissue Regeneration by Targeting The 5-HT2B Receptor in Chronic Liver Disease. Nature Medicine 17, 1668-73 (2011). The large number of pharmaceutical agents that block or stimulate the various 5-HT receptors is also indicative of the wide range of medical disorders that have been associated with 5-HT dysregulation (see for example: Wacker, D. et al., Structural Features for Functional Selectivity at Serotonin Receptors, Science (New York N Y.) 340, 615-9 (2013).

The rate-limiting step in 5-HT biosynthesis is the hydroxylation of tryptophan by dioxygen, which is catalyzed by tryptophan hydroxylase (TPH; EC 1.14.16.4) in the presence of the cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4). The resulting oxidized product, 5-hydroxy tryptophan (5-HTT) is subsequently decarboxylated by an aromatic amino acid decarboxylase (AAAD; EC 4.1.1.28) to produce 5-HT. Together with phenylalanine hydroxylase (PheOH) and tyrosine hydroxylase (TH), TPH belongs to the pterin-dependent aromatic amino acid hydroxylase family.

Two vertebrate isoforms of TPH, namely TPH1 and TPH2, have been identified. TPH1 is primarily expressed in the pineal gland and non-neuronal tissues, such as entero chromaffin (EC) cells located in the gastrointestinal (GI) tract. TPH2 (the dominant form in the brain) is expressed exclusively in neuronal cells, such as dorsal raphe or myenteric plexus cells. The peripheral and central systems involved in 5-HT biosynthesis are isolated, with 5-HT being unable to cross the blood-brain barrier. Therefore, the peripheral pharmacological effects of 5-HT can be modulated by agents affecting TPH in the periphery, mainly TPH1 in the gut.

A small number of phenylalanine-derived TPH1 inhibitors are known. One example, p-chlorophenylalanine (pCPA), a very weak and unselective irreversible inhibitor of TPH, has proven effective in treating chemotherapy-induced emesis, as well as diarrhea, in carcinoid tumor patients. However, pCPA is distributed centrally and, as a result, its administration has been linked to the onset of depression and other alterations of CNS functions in patients and animals. p-Ethynyl phenylalanine is a more selective and more potent TPH inhibitor than pCPA (Stokes, A, H., et al. p-Ethynylphenylalanine: A Potent Inhibitor of Tryptophan Hydroxylase. Journal of Neurochemistry 74, 2067-73 (2000), but also affects central 5-HT production and, like pCPA, is believed to irreversibly interfere with the synthetic behavior of TPH (and possibly interact with other proteins).

More recently, bulkier phenylalanine-derived TPH inhibitors have been reported to reduce intestinal 5-HT concentration without affecting brain 5-HT levels (Zhong, H. et al., Molecular dynamics simulation of tryptophan hydroxylase-1: binding modes and free energy analysis to phenylalanine derivative inhibitors. International Journal of Molecular Sciences 14, 9947-62 (2013); Ouyang, L., et al., Combined Structure-Based Pharmacophore and 3D-QSA Studies on Phenylalanine Series Compounds as TPH1 Inhibitors. International Journal of Molecular Sciences 13, 5348-63 (2012); Camilleri, M. LX-1031, A Tryptophan 5-hydroxylase Inhibitor, and its Potential in Chronic Diarrhea Associated with Increased Serotonin. Neurogastroenterology and Motility: The Official Journal of the European Gastrointestinal Motility Society 23, 193-200 (2011); Cianchetta, G., et al., Mechanism of Inhibition of Novel Tryptophan Hydroxylase Inhibitors Revealed by Co-crystal Structures and Kinetic Analysis. Current Chemical Genomics 4, 19-26 (2010); Jin, H., et al., Substituted 3-(4-(l,3,5-triazin-2-yl)-phenyl)-2-aminopropanoic Acids as Novel Tryptophan Hydroxylase Inhibitors. Bioorganic & Medicinal Chemistry Letters 19, 5229-32 (2009); Shi, Z.C., et al., Modulation of Peripheral Serotonin Levels by Novel Tryptophan Hydroxylase Inhibitors for the Potential Treatment of Functional Gastrointestinal Disorders. Journal of Medicinal Chemistry 51, 3684-7 (2008); Liu, Q., et al., Discovery and Characterization of Novel Tryptophan Hydroxylase Inhibitors That Selectively Inhibit Serotonin Synthesis in the Gastrointestinal Tract. The Journal of Pharmacology and Experimental Therapeutics 325, 47-55 (2008).

Spirocyclic compounds that act as inhibitors of THP and are useful in the treatment of various diseases and disorders associated with peripheral serotonin, including the cardiovascular diseases of pulmonary arterial hypertension (PAH) and associated pulmonary arterial hypertension (APAH) and carcinoid syndrome are known. (S)-ethyl 8-(2-amino-6-((R)-1-(5-chloro-[1,1′-biphenyl]-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylate and (S)-8-(2-amino-6-((R)-1-(5-chloro-[1,1T-biphenyl]-2-yl)-2,2,2-trifluoroeth-oxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylic acid are known compounds.

There is a need to selectively reduce tissue 5-HT (particularly intestinal 5-HT and lung 5-HT) levels as a means for treating and preventing 5-HT-associated diseases and modulation and/or reduction of serotonin levels, particularly peripheral serotonin levels, as well as 5-HIAA levels in the urine. There is also a need to achieve desirable AUC_(0-tau) levels in the bloodstream. There is a more particular need to treat or prevent the cardiovascular diseases of pulmonary arterial hypertension (PAH) and associated pulmonary arterial hypertension (APAH).

SUMMARY OF THE DISCLOSURE

According to the present disclosure, there is provided a daily dosage regimen for treating pulmonary arterial hypertension. The regimen takes the form of two discrete dosage forms. Each dosage form includes an amount from about 600 mg to about 800 mg of (S)-ethyl 8-(2-amino-6-((R)-1-(5-chloro-[1,1′-biphenyl]-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylate.

Further according to the present disclosure, there is provided a daily dosage regimen for treating pulmonary arterial hypertension. The regimen has an amount from about 1200 mg to about 1600 mg of (S)-ethyl 8-(2-amino-6-((R)-1-(5-chloro-[1,1′-biphenyl]-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]deca ne-3-carboxylate.

Also according to the present disclosure, there is provided a method for treating pulmonary arterial hypertension. The method has the step of administering to a human patient in need thereof an amount from about 1200 mg to about 1600 mg of (S)-ethyl 8-(2-amino-6-((R)-1-(5-chloro-[1,1′-biphenyl]-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylate per day.

Further according to the present disclosure, there is provided a method for reducing the level of serotonin biosynthesis by at least 50% in a human patient in need thereof within 14 days after commencement of treatment. The method has the step of administering to the human patient an amount from about 800 mg to about 1600 mg of (S)-ethyl 8-(2-amino-6-((R)-1-(5-chloro-[1,1′-biphenyl]-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylate per day.

Still further according to the present disclosure, there is provided a method for achieving an AUC_(0-tau) of ≥2530 ng.hr/mL of (S)-8-(2-amino-6-((R)-1-(5-chloro-[1,1′-biphenyl]-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylic acid in a human patient within 14 days after commencement of administration. The method has the step of administering daily to the human patient an effective amount of (S)-ethyl 8-(2-amino-6-((R)-1-(5-chloro-[1,1′-biphenyl]-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylate.

According to the present disclosure, there is provided a method of achieving a >50% reduction in urinary 5-HIAA in a human patient within 14 days after commencement of administration. The method has the step of administering daily to a human patient an effective amount of (S)-ethyl 8-(2-amino-6-((R)-1-(5-chloro-[1,1′-biphenyl]-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylate.

Further according to the present disclosure, there is provided a method for treating pulmonary arterial hypertension. The method has the step of administering daily to a human patient in need thereof an amount of (S)-ethyl 8-(2-amino-6-((R)-1-(5-chloro-[1,1′-biphenyl]-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylate sufficient to achieve an AUC_(0-tau) of ≥2530 ng.hr/mL of (S)-8-(2-amino-6-((R)-1-(5-chloro-[1,1′-biphenyl]-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylic acid within 14 days after commencement of administration.

According to the present disclosure, there is provided a method for treating pulmonary arterial hypertension. The method has the step of administering daily to a human patient in need thereof an amount of (S)-ethyl 8-(2-amino-6-((R)-1-(5-chloro-[1,1′-biphenyl]-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylate sufficient to achieve a >50% reduction in urinary 5-HIAA within 14 days after commencement of administration.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present disclosure are described herein with reference to the following figures.

FIG. 1 is a plot of an XRPD of a crystalline compound of (S)-ethyl 8-(2-amino-6-((R)-1-(5-chloro-[1,1′-biphenyl]-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylate according to the present disclosure (crystalline Form 3).

FIG. 2 is a plot of an XRPD of a crystalline compound of (S)-ethyl 8-(2-amino-6-((R)-1-(5-chloro-[1,1′-biphenyl]-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylate of a different polymorphic form than that of FIG. 1 (crystalline Form 1).

FIG. 3 a is a plot of mean (±SD) plasma concentration-time profiles of KAR5585 following administration of 100-2000 mg of KAR5585 under fasting conditions—Cohorts 1-6 (linear scale).

FIG. 3 b is a plot of mean (±SD) plasma concentration-time profiles of KAR5417 following administration of 100-2000 mg of KAR5585 under fasting conditions—Cohorts 1-6 (linear scale) for KAR5417.

FIG. 4 a is a plot of mean (±SD) plasma concentration-time profiles of KAR5585 and KAR5417 following administration of 100-2000 mg of KAR5585 under fasting conditions—Cohorts 1-6 (log scale) for KAR5585.

FIG. 4 b is a plot of mean (±SD) plasma concentration-time profiles of KAR5585 and KAR5417 following administration of 100-2000 mg of KAR5585 under fasting conditions—Cohorts 1-6 (log scale) for KAR5417.

FIG. 5 a is a plot of dose vs. plasma C_(max) values for KAR5585 and KAR5417 in fasted healthy volunteers following a single oral dose of KAR5585.

FIG. 5 b is a plot of dose vs. plasma C_(max) values for KAR5585 and KAR5417 in fasted healthy volunteers following a single oral dose of KAR5417.

FIG. 6 a is a plot of dose vs. plasma AUC_(inf) values for KAR5585 and KAR5417 in fasted healthy volunteers following a single oral dose of KAR5585.

FIG. 6 b is a plot of dose vs. plasma AUC_(inf) values for KAR5585 and KAR5417 in fasted healthy volunteers following a single oral dose of KAR5585.

FIG. 7 is a bar graph of mean (±SD) total percent of dose excreted in urine as KAR5417 in fasted healthy volunteers following a single oral dose of KAR5585.

FIG. 8 is a plot of cumulative mean (±SD) urine recoveries vs. sampling time of KAR5417 in fasted healthy volunteers following a single oral dose of KAR5585.

FIG. 9 a is a plot of mean concentration vs. time profiles of KAR5585 in fasted and fed healthy volunteers following BID oral administration of KAR5585 for 14 days (semi-log scale)

FIG. 9 b is a plot of mean concentration vs. time profiles of KAR5417 in fasted and fed healthy volunteers following BID oral administration of KAR5585 for 14 days (semi-log scale)

FIG. 10 is a plot of mean (±SD) concentration vs. time profiles of KAR5585 and KAR5417 in fasted healthy volunteers following BID oral administration of 100 mg KAR5585 for 14 days.

FIG. 11 is a plot of mean (±SD) concentration vs. time profiles of KAR5585 and KAR5417 in fed healthy volunteers following BID oral administration of 100 mg KAR5585 for 14 days.

FIG. 12 is a plot of mean (±SD) concentration vs. time profiles of KAR5585 and KAR5417 in fed healthy volunteers following BID oral administration of 200 mg KAR5585 for 14 days.

FIG. 13 is a plot of mean (±SD) concentration vs. time profiles of KAR5585 and KAR5417 in fed healthy volunteers following BID oral administration of 400 mg KAR5585 for 14 days.

FIG. 14 a is a plot of dose vs. C_(max) for KAR5585 in fed healthy volunteers following BID oral administration of KAR5585 for 14 days.

FIG. 14 b is a plot of dose vs. C_(max) for KAR5417 in fed healthy volunteers following BID oral administration of KAR5585 for 14 days.

FIG. 15 a is a plot of dose vs. C_(max) for KAR5585 in fed healthy volunteers following BID oral administration of KAR5585 for 14 days.

FIG. 15 b is a plot of dose vs. C_(max) for KAR5417 in fed healthy volunteers following BID oral administration of KAR5585 for 14 days.

FIG. 16 a is a plot of dose vs. AUC₀₋₁₂ for KAR5585 in fed healthy volunteers following BID oral administration of KAR5585 for 14 days.

FIG. 16 b is a plot of dose vs. AUC₀₋₁₂ for KAR5417 in fed healthy volunteers following BID oral administration of KAR5585 for 14 days.

FIG. 17 a is a plot of dose vs. AUC₀₋₁₂ for KAR5585 in fed healthy volunteers following BID oral administration of KAR5585 for 14 days.

FIG. 17 b is a plot of dose vs. AUC₀₋₁₂ for KAR5417 in fed healthy volunteers following BID oral administration of KAR5585 for 14 days.

FIG. 18 a is a plot of mean (±SD) plasma concentration-time profiles of KAR5585 following administration of 400 mg of KAR5585 under fed and fasting conditions—Cohorts 3A and 3B (linear scale).

FIG. 18 b is a plot of mean (±SD) plasma concentration-time profiles of KAR5417 following administration of 400 mg of KAR5585 under fed and fasting conditions—Cohorts 3A and 3B (linear scale).

FIG. 19 a is a plot of mean (±SD) plasma concentration-time profiles of KAR5585 following administration of 400 mg of KAR5585 under fed and fasting conditions—Cohorts 3A and 3B (log scale).

FIG. 19 b is a plot of mean (±SD) plasma concentration-time profiles of KAR5417 following administration of 400 mg of KAR5585 under fed and fasting conditions—Cohorts 3A and 3B (log scale).

FIG. 20 is a plot of relative change from baseline percentage of plasma 5-HIAA vs AUC₀₋₂₄ at Day 14.

DETAILED DESCRIPTION OF THE DISCLOSURE

(S)-ethyl 8-(2-amino-6-((R)-1-(5-chloro-[1,1′-biphenyl]-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylate (“KAR5585”) is a useful TPH1 inhibitor. The amorphous form of KAR5585 is disclosed at Example 63i of U.S. Pat. No. 9,199,994. KAR5585 has the following structure:

Two crystalline forms of KAR5585, i.e., polymorphs are known. One is denoted as “crystalline Form 3” and the other as “crystalline Form 1”. Form 3 exhibits substantially greater thermodynamic stability compared to Form 1 and is supportive of longer shelf life, particularly at temperatures of less than 95° C. and more particularly at temperatures of less than 80° C.

The crystalline Form 3 exhibits the XRPD (X-ray powder diffraction) pattern set forth below in Table 1.

TABLE 1 Observed Peaks for X-ray Powder Diffraction Pattern for KAR5585, Form 3 Peak position (°2θ) d space (Å) Intensity (%)  8.78 ± 0.20 10.077 ± 0.235  90 12.00 ± 0.20 7.375 ± 0.125 25 13.47 ± 0.20 6.573 ± 0.099 39 14.02 ± 0.20 6.316 ± 0.091 12 14.87 ± 0.20 5.956 ± 0.081 71 15.39 ± 0.20 5.757 ± 0.075 72 15.61 ± 0.20 5.677 ± 0.073 78 15.89 ± 0.20 5.576 ± 0.071 50 16.31 ± 0.20 5.434 ± 0.067 7 17.70 ± 0.20 5.011 ± 0.057 34 18.45 ± 0.20 4.809 ± 0.052 70 19.05 ± 0.20 4.658 ± 0.049 100 20.12 ± 0.20 4.413 ± 0.044 42 20.57 ± 0.20 4.317 ± 0.042 68 20.84 ± 0.20 4.262 ± 0.041 39 21.46 ± 0.20 4.141 ± 0.039 49 21.94 ± 0.20 4.051 ± 0.037 18 22.56 ± 0.20 3.941 ± 0.035 31 22.90 ± 0.20 3.884 ± 0.034 17 23.90 ± 0.20 3.723 ± 0.031 35 24.32 ± 0.20 3.660 ± 0.030 13 25.07 ± 0.20 3.552 ± 0.028 12 26.54 ± 0.20 3.359 ± 0.025 17 26.76 ± 0.20 3.332 ± 0.025 18 27.79 ± 0.20 3.210 ± 0.023 8 28.21 ± 0.20 3.163 ± 0.022 19 29.48 ± 0.20 3.030 ± 0.020 9

In another aspect, Form 3 exhibits prominent XRPD peaks set forth below in Table 2.

TABLE 2 Prominent Observed Peaks for X-ray Powder Diffraction Pattern for KAR5585, Form 3 Peak position (°2θ) d space (Å) Intensity (%)  8.78 ± 0.20 10.077 ± 0.235  90 14.87 ± 0.20 5.956 ± 0.081 71 15.39 ± 0.20 5.757 ± 0.075 72 15.61 ± 0.20 5.677 ± 0.073 78 18.45 ± 0.20 4.809 ± 0.052 70 19.05 ± 0.20 4.658 ± 0.049 100

In yet another aspect, Form 3 exhibits a characteristic XRPD peak at 19.05±0.20 (°2θ).

Crystalline Form 1 crystalline compound exhibits the XRPD (X-ray powder diffraction) pattern set forth below in Table 3.

TABLE 3 Observed Peaks for X-Ray Powder Diffraction Pattern for Form 1 Peak position (°2θ) d space (Å) Intensity (%)  5.92 ± 0.20 14.936 ± 0.522  27  9.01 ± 0.20 9.816 ± 0.222 11  9.68 ± 0.20 9.140 ± 0.192 9 10.38 ± 0.20 8.523 ± 0.167 9 10.95 ± 0.20 8.082 ± 0.150 30 11.85 ± 0.20 7.468 ± 0.128 6 12.90 ± 0.20 6.861 ± 0.108 43 13.89 ± 0.20 6.376 ± 0.093 65 14.62 ± 0.20 6.057 ± 0.084 31 15.04 ± 0.20 5.890 ± 0.079 44 15.41 ± 0.20 5.750 ± 0.075 38 17.13 ± 0.20 5.176 ± 0.061 30 17.83 ± 0.20 4.974 ± 0.056 37 18.72 ± 0.20 4.741 ± 0.051 14 19.44 ± 0.20 4.567 ± 0.047 100 19.79 ± 0.20 4.487 ± 0.045 30 20.11 ± 0.20 4.417 ± 0.044 97 20.34 ± 0.20 4.366 ± 0.043 44 20.84 ± 0.20 4.262 ± 0.041 14 21.41 ± 0.20 4.151 ± 0.039 10 21.88 ± 0.20 4.063 ± 0.037 11 22.28 ± 0.20 3.991 ± 0.036 25 22.83 ± 0.20 3.895 ± 0.034 60 23.85 ± 0.20 3.731 ± 0.031 13 24.40 ± 0.20 3.648 ± 0.030 9 25.45 ± 0.20 3.500 ± 0.027 9 25.97 ± 0.20 3.431 ± 0.026 12 27.22 ± 0.20 3.276 ± 0.024 15 27.58 ± 0.20 3.235 ± 0.023 23 28.06 ± 0.20 3.180 ± 0.022 12 28.66 ± 0.20 3.115 ± 0.021 7

In still another aspect, Form 1 exhibits prominent XRPD peaks set forth below in Table 4.

TABLE 4 Prominent Observed Peaks for X-Ray Powder Diffraction Pattern for Compound 1, Form 1 Peak position (°2θ) d space (Å) Intensity (%) 12.90 ± 0.20 6.861 ± 0.108 43 13.89 ± 0.20 6.376 ± 0.093 65 15.04 ± 0.20 5.890 ± 0.079 44 19.44 ± 0.20 4.567 ± 0.047 100 20.11 ± 0.20 4.417 ± 0.044 97 20.34 ± 0.20 4.366 ± 0.043 44 22.83 ± 0.20 3.895 ± 0.034 60

The amorphous form of (S)-ethyl 8-(2-amino-6-((R)-1-(5-chloro-[1,1′-biphenyl]-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylate can be prepared by the method set forth in Example 63i of U.S. Pat. No. 9,199,994, which is incorporated herein by reference in its entirety. The amorphous form can then be converted to crystalline form by methods described in U.S. Ser. No. 16/683,509, filed Nov. 14, 2019, which is incorporated herein by reference in its entirety. Forms 1 and 3 can be prepared by the methods set forth in U.S. Ser. No. 16/683,509.

The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The Form 3 compound can be used to inhibit the activity of the TPH1 enzyme in a cell by contacting the cell with an inhibiting amount of a compound of the disclosure. The cell can be part of the tissue of a living organism, or can be in culture, or isolated from a living organism. Additionally, the Form 3 compound can be used to inhibit the activity of the TPH1 enzyme in an animal, individual, or patient, by administering an inhibiting amount of a compound of the disclosure to the cell, animal, individual, or human patient.

The Form 3 compound can also lower peripheral serotonin levels in an animal, individual, or patient, by administering an effective amount of a compound of the disclosure to the animal, individual, or patient. In some embodiments, the Form 3 compound can lower levels of peripheral serotonin (e.g., 5-HT in the GI tract or lung tissue) selectively over non-peripheral serotonin (e.g., 5-HT in the CNS). In some embodiments, the selectivity can be 2-fold or more, 3-fold or more, 5-fold or more, 10-fold or more, 50-fold or more, or 100-fold or more.

As TPH1 inhibitors that can lower peripheral serotonin levels, the Form 3 compound is useful in the treatment and prevention of various diseases associated with abnormal expression or activity of the TPH1 enzyme, or diseases associated with elevated or abnormal peripheral serotonin levels. In some embodiments, the treatment or prevention includes administering to a patient in need thereof a therapeutically effective amount of a TPH1 inhibitor of the Form 3 compound. The Form 3 compound is also useful in the treatment and prevention of serotonin syndrome.

The efficacy of amorphous (S)-ethyl 8-(2-amino-6-((R)-1-(5-chloro-[1,1′-biphenyl]-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylate in inhibiting TPH1 in mice was demonstrated in U.S. Pat. No. 9,199,994 in biological assays at Example 63i and Table 27.

Biological assays, some of which are described herein, can be used to determine the inhibitory effect of compounds against TPH (such as TPH1) in vitro and/or in vivo. In vitro biochemical assays for human, mouse, and rat TPH1 and human TPH2, PheOH, and TH may be used to measure inhibition of enzyme activity and the selectivity among TPH1, TPH2, PheOH, and TH. In addition, the efficacy of these compounds can be determined, for example, by measuring their effect on intestinal 5-HT levels in rodents after oral administration.

The metabolite of KAR5585 is (S)-8-(2-amino-6-((R)-1-(5-chloro-[1,1T-biphenyl]-2-yl)-2,2,2-trifluoroeth-oxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylic acid, which is of the formula

(S)-8-(2-amino-6-((R)-1-(5-chloro-[1,1T-biphenyl]-2-yl)-2,2,2-trifluoroeth-oxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylic acid is referred to herein as KAR5417. When KAR5585 enters the bloodstream, it substantially converts to KAR5417. The amorphous form of KAR5417 can be prepared by the method set forth in Example 34c of U.S. Pat. No. 9,199,994.

KAR5585 has been found to be particularly useful in treating and preventing pulmonary arterial hypertension (PAH). Prior studies have characterized treatment of PAH in rats, but there remains a need for efficacy and dosage to be characterized such that effective treatment of human adults can be carried out.

One aspect of the present disclosure is a daily dosage regimen for treatment of PAH in adults. The regimen takes the form of two discrete dosage forms. Each dosage form includes an amount from about 600 mg to about 800 mg of (S)-ethyl 8-(2-amino-6-((R)-1-(5-chloro-[1,1′-biphenyl]-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylate of Form 3. A preferred daily regimen employs two oral dosage forms taken twice per day (BID) up to 14 days. Another preferred aspect are dosage forms having 600 mg or 800 mg of KAR5585 (Form 3).

Another aspect of the dosage regimen for treating pulmonary arterial hypertension is administration of an amount from about 1200 mg to about 1600 mg of (S)-ethyl 8-(2-amino-6-((R)-1-(5-chloro-[1,1′-biphenyl]-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylate per day.

Another aspect of the present disclosure is a method for reducing the level of serotonin (5-HT) biosynthesis by at least 50% in a human patient within 14 days after commencement of treatment. The method has the step of administering to the human patient about 800 mg to about 1600 mg of (S)-ethyl 8-(2-amino-6-((R)-1-(5-chloro-[1,1′-biphenyl]-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylate per day. Serotonin levels are determined by the methods disclosed in the examples below.

Another aspect of the present disclosure is treatment of pulmonary arterial hypertension by achievement an AUC_(0-tau) of ≥2530 ng.hr/mL of (S)-8-(2-amino-6-((R)-1-(5-chloro-[1,1′-biphenyl]-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylic acid in a human patient within 14 days after commencement of administration of a sufficient amount to the human patient. A preferred level of AUC_(0-tau) of ≥2530 ng.hr/mL. AUC_(0-tau) is determined by methods disclosed in the examples below.

Another aspect of the present disclosure is treatment of pulmonary arterial hypertension by achievement of a >50% reduction in urinary 5-HIAA in a human patient within 14 days after commencement of administration of a sufficient amount to the human patient. Urinary 5-HIAA is determined by methods disclosed in the examples below.

Other diseases treatable or preventable by administering a TPH1 inhibitor of the disclosure include bone disease such as, for example, osteoporosis, osteoporosis pseudoglioma syndrome (OPPG), osteopenia, osteomalacia, renal osteodystrophy, Paget's disease, fractures, and bone metastasis, In some embodiments, the disease is osteoporosis, such as primary type 1 (e.g., postmenopausal osteoporosis), primary type 2 (e.g., senile osteoporosis), and secondary (e.g., steroid- or glucocorticoid-induced osteoporosis).

Further diseases treatable or preventable by the methods of the disclosure include cardiovascular diseases such as atherosclerosis and pulmonary hypertension (PH), including idiopathic or familial PH, and including PH associated with or brought on by other diseases or conditions. In some embodiments, the PH disease is pulmonary arterial hypertension (PAH).

The types of PAH treatable according to the methods of the disclosure include (1) idiopathic (IPAH), (2) familial (FPAH), and (3) associated (APAH) which is the most common type of PAH. The latter is PAH which is associated with other medical conditions including, for example, (1) collagen vascular disease (or connective tissue disease) that include autoimmune diseases such as scleroderma or lupus; (2) congenital heart and lung disease; (3) portal hypertension (e.g., resulting from liver disease); (4) HIV infection; (5) drugs (e.g., appetite suppressants, cocaine, and amphetamines; and (6) other conditions including thyroid disorders, glycogen storage disease, Gaucher disease, hereditary hemorrhagic telangiectasia, hemoglobinopathies, myeloproliferative disorders, and splenectomy. APAH can also be PAH associated with abnormal narrowing in the pulmonary veins and/or capillaries such as in pulmonary veno-occlusive disease (PVOD) and pulmonary capillary hemangiomatosis. Another type of PAH is associated with persistent pulmonary hypertension of the newborn (PPHN).

Further diseases treatable or preventable by the methods of the present disclosure include metabolic diseases such as diabetes and hyperlipidemia; pulmonary diseases such as chronic obstructive pulmonary disease (COPD), and pulmonary embolism; gastrointestinal diseases such as IBD, colitis, chemotherapy-induced emesis, diarrhea, carcinoid syndrome, celiac disease, Crohn's disease, abdominal pain, dyspepsia, constipation, lactose intolerance, MEN types I and II, Ogilvie's syndrome, pancreatic cholera syndrome, pancreatic insufficiency, pheochromacytoma, scleroderma, somatization disorder, Zollinger-Ellison Syndrome, or other gastrointestinal inflammatory conditions; liver diseases such as chronic liver disease; cancers such as liver cancer, breast cancer, cholangiocarcinoma, colon cancer, colorectal cancer, neuroendocrine tumors, pancreatic cancer, prostate cancer, and bone cancer (e.g., osteosarcoma, chrondrosarcoma, Ewings sarcoma, osteoblastoma, osteoid osteoma, osteochondroma, enchondroma, chondromyxoid fibroma, aneurysmal bone cyst, unicameral bone cyst, giant cell tumor, and bone tumors); blood diseases (e.g., myeloproliferative syndrome, myelodysplasia syndrome, Hodgkin's lymphoma, non-Hodgkin's lymphoma, myeloma, and anemia such as aplastic anemia and anemia associated with kidney disease; and blood cancers (e.g., leukemias such as acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), and chronic myeloid leukemia (CML)).

A further treatable disease is the treatment and prevention of carcinoid syndrome. Carcinoid syndrome is a paraneoplastic syndrome exhibiting the signs and symptoms that occur secondary to carcinoid tumors. Carcinoid syndrome is caused by a carcinoid tumor that secretes serotonin or other hormones into the bloodstream. Carcinoid tumors usually occur in the gastrointestinal tract, including the stomach, appendix, small intestine, colon, and rectum or in the lungs. Common symptoms include skin flushing, facial skin lesions, diarrhea, irritable bowel syndrome, cramping, difficulty breathing, and rapid heartbeat.

In some embodiments, the present disclosure includes methods of lowering plasma cholesterol, lowering plasma triglycerides, lowering plasma glycerol, lowering plasma free fatty acids in a patient by administering to said patient a therapeutically effective amount of a compound of the disclosure.

KAR5585 is also useful in the treatment and prevention of inflammatory disease, such as allergic airway inflammation (e.g., asthma).

As used herein, the term “cell” is meant to refer to a cell that is in vitro, ex vivo or in vivo. In some embodiments, an ex vivo cell can be part of a tissue sample excised from an organism such as a mammal. In some embodiments, an in vitro cell can be a cell in a cell culture. In some embodiments, an in vivo cell is a cell living in an organism such as a mammal.

As used herein, the term “contacting” refers to the bringing together of indicated moieties in an in vitro system or an in vivo system. For example, “contacting” the enzyme with a compound of the disclosure includes the administration of a compound of the present disclosure to an individual or patient, such as a human, having the TPH1 enzyme, as well as, for example, introducing a compound of the disclosure into a sample containing a cellular or purified preparation containing the TPH1 enzyme.

As used herein, the term “individual” or “patient” used interchangeably, refers to any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and, most preferably, humans.

As used herein, the phrase “therapeutically effective amount” refers to the amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue, system, animal, individual or human that is being sought by a researcher, veterinarian, medical doctor or other clinician.

As used herein, the term “treating” or “treatment” refers to 1) inhibiting the disease; for example, inhibiting a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., arresting further development of the pathology and/or symptomatology) or 2) ameliorating the disease; for example, ameliorating a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., reversing the pathology and/or symptomatology).

As used herein, the term “preventing” or “prevention” refers to inhibiting onset or worsening of the disease; for example, in an individual who may be predisposed to the disease, condition or disorder but does not yet experience or display the pathology or symptomatology of the disease.

KAR5585 can be administered to human patients in need of such treatment in appropriate dosages that will provide prophylactic and/or therapeutic efficacy. The dose required for use in the treatment or prevention of any particular disease or disorder will typically vary from patient to patient depending on, for example, particular compound or composition selected, the route of administration, the nature of the condition being treated, the age and condition of the patient, concurrent medication or special diets then being followed by the patient, and other factors. The appropriate dosage can be determined by the treating physician.

KAR5585 can be administered orally, subcutaneously, topically, parenterally, by inhalation spray or rectally in dosage unit formulations containing pharmaceutically acceptable carriers, adjuvants and vehicles. Parenteral administration can involve subcutaneous injections, intravenous or intramuscular injections or infusion techniques. Treatment duration can be as long as deemed necessary by a treating physician. The compositions can be administered one to four or more times per day. A treatment period can terminate when a desired result, for example, a particular therapeutic effect, is achieved. Alternatively, a treatment period can be continued indefinitely.

In some embodiments, the pharmaceutical compositions can be prepared as solid dosage forms for oral administration (e.g., capsules, tablets, pills, dragees, powders, granules and the like). A tablet can be prepared by compression or molding. Compressed tablets can include one or more binders, lubricants, glidants, inert diluents, preservatives, disintegrants, or dispersing agents. Tablets and other solid dosage forms, such as capsules, pills and granules, can include coatings, such as enteric coatings.

Solid and liquid dosage forms can be formulated so that they conform to a desired release profile, e.g., immediate release, delayed release, and extended or sustained release.

The amount of KAR5585 to be administered will vary depending on factors such as the following: method of administration, release profile, and composition formulation. Typically, for KAR5585 in an oral dosage form to treat or prevent a disease, particularly PH/PAH/APAH/IPAH/FPAH, a typical dosage will be about 1 mg/kg/day to about 50 mg/kg/day and more typically from about 5 mg/kg/day to about 30 mg/kg/day, based on the weight of the patient. A most preferred active is Form 3. Individual oral dosage forms typically have from about 50 mg to about 3000 mg of KAR5585 and additional amounts of one or more pharmaceutically acceptable excipients. Other useful individual oral dosage forms can, by way of example, have KAR5585 in amounts of 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, or 400 mg, 450 mg, 500 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, 800 mg, 900 mg, 950 mg, 1000 mg, 1050 mg, 1100 mg, 1150 mg, and about 1200 mg. Other amounts between 50 mg to 3000 mg are possible, for example, from about 325 mg to about 475 mg, from about 350 mg to about 500 mg, from about 375 to about 525 mg, from about 400 mg to about 550 mg, from about 425 mg to about 575 mg, from about 450 mg to about 600 mg, from about 475 mg to about 625 mg, from about 500 mg to about 650 mg, from about 525 mg to about 675 mg, from about 550 mg to about 700 mg, from about 575 mg to about 725 mg, from about 600 mg to about 750 mg, from about 625 mg to about 775 mg, from about 650 mg to about 800 mg, from about 675 mg to about 825 mg, from about 700 mg to about 850 mg, from about 725 mg to about 875 mg, from about 750 mg to about 900 mg, from about 775 mg to about 925 mg, from about 800 mg to about 950 mg, from about 825 to about 975, from about 850 mg to about 1000 mg, from about 900 mg to about 1150 mg, from about 1000 mg to about 1150 mg, from about 1100 mg to about 1250 mg, from about 1200 mg to about 1350 mg, and from about 1200 mg to about 1350 mg. Particularly preferred dosage regimens are 600 mg to 800 mg twice per day (BID). Especially preferred regimen embodiments are 600 mg BID and 800 mg BID.

“wt %” means weight percent based on the total weight of the composition or formulation.

Preferred dosage forms have the crystalline compound of Form 3 present in a proportion that is 90 wt % or more, and more preferably 95 wt % or more, by weight of any (S)-ethyl 8-(2-amino-6-((R)-1-(5-chloro-[1,1′-biphenyl]-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylate present.

Dosage forms have the crystalline compound of Form 3 therein in any amount or proportion. Typical proportions include about 20 wt % or more, about 60 wt % or more, and about 90 wt % or more, based on the total weight of the dosage form (with the balance predominantly excipients, carriers, and vehicles). Particularly useful proportions are 25 wt % and 60 wt %.

Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents, or mixtures thereof, and powders. Liquid dosage forms for oral administration can include, for example, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. Suspensions can include one or more suspending agents.

Dosage forms for transdermal administration of a subject composition include powders, sprays, ointments, pastes, creams, lotions, gels, solutions and patches.

The Form 3 compound and compositions containing same can be administered in the form of an aerosol, which can be administered, for example, by a sonic nebulizer.

Pharmaceutical compositions suitable for parenteral administration can include the Form 3 compound together with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions.

Alternatively, the composition can be in the form of a sterile powder which can be reconstituted into a sterile injectable solutions or dispersion just prior to use.

The disclosure is further illustrated herein by the following non-limiting examples.

EXAMPLES

TABLE 5 Abbreviations used in the examples include the following: Abbreviation Explanation % CV percent coefficient of variation β-hCG beta human chorionic gonadotropin 5-HIAA 5-hydroxyindoleacetic acid 5-HT 5-hydroxytryptamine (serotonin) Adjusted urine 5-HIAA the 5-HIAA measured in urine during 24 hours, adjusted for the mean 24- hour creatinine excretion on Days 1, 7, and 14 AE adverse event AESI adverse event of special interest ALP alkaline phosphatase ALT alanine aminotransferase AST aspartate aminotransferase AUC area under the concentration versus time curve AUC₀₋₁₂ area under the concentration versus time curve from time 0 to 12 hours after dosing, computed using the trapezoidal rule AUC₀₋₂₄ area under the concentration versus time curve from time 0 to 24 hours after dosing, computed using the trapezoidal rule AUC_(0-inf) area under the concentration versus time curve from time 0 extrapolated to infinity AUC_(0-tau) area under the concentration-time curve from time 0 to time of last quantifiable concentration BID twice daily (from Latin, bis in die) BILI bilirubin BLQ below the lower limit of quantitation BMI body mass index BP blood pressure C_(12 hr) concentration, obtained 12 hr after an administered dose C_(max) maximum (or peak) serum concentration C_(min) minimum serum concentration CI confidence interval CL/F apparent oral clearance CRO contract research organization; Ce3, Inc. CRU clinical research unit ddHR placebo-adjusted mean change from Baseline in heart rate ddQTcF placebo-adjusted mean change from Baseline in QTcF ECG electrocardiogram eCRF electronic case report form Estimated urine 5-HIAA the 5-HIAA measured in urine during 24 hours, corrected for the estimated creatinine excretion based on subject weight, age, gender, and race FDA Food and Drug Administration GCP Good Clinical Practice GGT gamma-glutamyl transpeptidase Geo. geometric Hg millimeters of mercury HR heart rate ICF informed consent form ICH International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use IRB Institutional Review Board IUD intrauterine device KAR5417 an active metabolite of the investigational product KAR5585 KAR5585 the investigational product; it is a prodrug of KAR5417 LDH lactate dehydrogenase Max maximum Measured urine 5-HIAA the 5-HIAA measured in urine during 24 hours, without adjustment for daily variations in creatinine excretion MedDRA Medical Dictionary for Regulatory Activities Min minimum MOA mechanism of action N, n number OAE other significant adverse event OTC over the counter PAH pulmonary arterial hypertension Param. parameter PCS potentially clinically significant PD pharmacodynamics(s) PI Principal Investigator; the investigator who leads the study conduct at an individual study center. Every study center has a PI. PK pharmacokinetic(s) PT preferred term QD once daily (from Latin, quaque die) QTcF QT interval corrected for heart rate by applying Fridericia's formula; the unit for QTcF is milliseconds (msec) R_(AUC0-12) accumulation ratio for AUC₀₋₁₂ R_(Cmax) accumulation ratio for C_(max) SAE serious adverse event SAP Statistical Analysis Plan SD standard deviation SOC system organ class Stat. statistical SRC Safety Review Committee Study drug investigational product, reference product (placebo), or both t_(1/2) apparent terminal half-life after oral administration TEAE treatment-emergent adverse event t_(max) time of maximum observed concentration TPH1 tryptophan hydroxylase 1 Urine 5-HIAA/24 urine 5-HIAA/24 hours divided by corresponding total creatinine hours/g creatinine (gram) measured/24 hours WHODD World Health Organization Drug Dictionary

Methodology

Study KAR5585-101 was a first-in-human, Phase 1, randomized, double-blind, placebo-controlled, single-center trial conducted in 2 parts to assess the safety, tolerability, PK, cardiac conduction, and biomarkers of target engagement effects of single ascending doses (SAD, Part 1) and multiple ascending doses (MAD, Part 2) of KAR5585 in healthy adult subjects. Food effect was to be evaluated in Part 1, Period 2. Part 2 was permitted to begin before Part 1 was completed and, in each part, the data from each dose Cohort were to be reviewed for safety before the next dose Cohort was enrolled.

The KAR5585 administered doses were the following:

-   -   In Part 1 (SAD) Period 1 (fasting): 100 mg, 200 mg, 400 mg, 700         mg, 1200 mg, or 2000 mg or matching placebo in Cohorts 1 through         6, respectively     -   In Part 1 (SAD) Period 2 (fed, high-fat food effect): 400 mg in         Cohort 3 only     -   In Part 2 (MAD): 100 mg (fasting), 100 mg (fed), 200 mg (fed),         and 400 mg (fed) or matching placebo administered twice daily         (BID) approximately every 12 hours for 27 doses in Cohorts 1         through 4, respectively.     -   Doses of KAR5585 and placebo were to be administered orally in a         capsule dosage form.

Active drug capsules containing KAR5585 were provided in 50, 200, and 300 mg strengths; thus, more than 1 capsule was required to achieve some of the study doses.

Number of Patients (Planned and Analyzed):

Enrollment of 60 adult subjects (5 Cohorts of 12 subjects each) was originally planned in each part. In addition, up to 2 Cohorts of 12 subjects each may have been added to either or both parts, if necessary, based upon safety and PK data from the previous Cohorts, for a total of up to 168 subjects participating in the trial. Subjects were allowed to participate in Part 1 or Part 2, but not in both parts.

Part 1 had 6 Cohorts of 12 subjects each (for a total of 72 subjects) and Part 2 had 4 Cohorts of 12 subjects each (for a total of 48 subjects). In Part 1, the 12 subjects in Cohort 3 (400 mg)were studied twice, once under fasting conditions (Period 1) and once under fed conditions (Period 2) for food-effect assessment.

Test Product, Dose and Mode of Administration, Batch Number:

KAR5585, 50, 200 and 300 mg oral capsules administered as single doses of 100 mg, 200 mg, 400 mg, 700 mg, 1200 mg or 2000 mg (Part 1) or multiple doses of 100 mg, 200 mg or 400 mg every 12 hours for 14 days (27 doses) (Part 2). Batches were Batch PID-19JUL15-111 (50 mg), PID-19JUL15-110 (200 mg), PID-19JUL15-109 (300 mg)

Duration of Treatment:

Part 1 (SAD): Single dose under fasted conditions; subjects included in the fed Cohort were to receive a second dose of KAR5585 (same dose level) or placebo.

Part 2 (MAD): BID doses (separated by approximately 12 hours on Days 1 to 13) and a single AM dose on Day 14. One or more of the MAD Cohort could have been dosed once daily (QD).

Reference Therapy, Dose and Mode of Administration, Batch Number:

Placebo, oral capsules. Batch PID-19JUL15-108 (matching 50 mg); PID-19JUL15-107 (matching 200 mg and 300 mg)

Pharmacokinetics:

Pharmacokinetic characteristics of KAR5585 and KAR5417 were to be evaluated in Parts 1 and 2 by measuring drug concentrations in plasma and urine and calculating PK parameters. Biomarkers:

Biomarkers were to be evaluated in Part 2 by measuring concentrations of serotonin (5-hydroxytryptamine [5-HT]) in serum and 5-hydroxyindoleacetic acid (5-HIAA) in plasma and urine. As well, the relationship of biomarkers to PK parameters was to be evaluated.

Biomarker Analysis:

The biomarker analysis was to be based on the Biomarker Population (all subjects with evaluable biomarker measurements at baseline [Day 1 predose] and at least one after dosing [Day 7 or Day 14]). For analyses focused on the relationship between the PK parameters area under the concentration versus time curve (AUC) computed using the trapezoidal rule from time 0 to 24 hours after dosing (AUC₀₋₂₄), minimum observed concentration (C_(min)), and maximum observed concentration (C_(max)), and biomarkers, only subjects who had all biomarkers and these PK parameters assessed were to be included.

Biomarker data were to be collected only in Part 2. The following biomarkers were to be analyzed:

-   -   Serum 5-HT     -   Plasma 5-HIAA     -   Urine 5-HIAA/24 hours (as measured, adjusted, estimated, and per         gram creatinine)

The a priori primary biomarker endpoint was to be the change in plasma 5-HIAA concentration on Day 14 from Day 1. The null hypothesis to be tested was that there was no difference between the change from Day 1 predose to Day 14 between placebo and KAR5585.

Linear regression was to be performed using biomarker changes, absolute and relative (%), from Day 1, at predose on Day 7 or Day 14 versus AUC₀₋₂₄, C_(max), and C_(min) in corresponding study days. The slopes and corresponding 95% confidence intervals (CIs) were to be tabulated.

Summary statistics (mean, standard deviation [SD], median, minimum [Min], and maximum [Max]) were to be tabulated for each biomarker, by Cohort, with absolute and relative (%) changes from Day 1 predose. Corresponding time course plots of the mean measurement for each biomarker were to be displayed graphically. The time course for each individual subject, by Cohort, was also to be displayed graphically.

For each biomarker, least mean squared differences and the corresponding 95% CIs were to be estimated for change from baseline between KAR5585 treatment groups and placebo at Day 7 or Day 14. The values were to be tabulated with P values testing whether the difference of changes was equal to 0.

Absolute and relative changes in each biomarker at Day 7 and Day 14 from Day 1 predose versus AUC₀₋₂₄, C_(max), and C_(min) were to be displayed graphically.

Blood and urine sample collection details for biomarker analysis and the biomarker concentrations were to be listed.

Pharmacokinetic Analysis:

The PK analysis was to be based on the PK Concentration Population (all subjects receiving active study medication and having any measurable plasma concentration of study medication at any time point) and the PK Evaluable Population (all subjects with sufficient KAR5585 and KAR5417 concentration-time data to support PK analysis). Pharmacokinetic data were to be summarized descriptively.

Summary—Conclusions Pharmacokinetics Results: Part 1 SAD PK:

KAR5585 is a prodrug for the active tryptophan hydroxylate 1 (TPH1) inhibitor KAR5417. Following KAR5585 administration, KAR5585 was rapidly absorbed. The median time of maximum observed concentration (t_(max)) ranged between 1.5 and 3 hours postdose. Following administration of KAR5585, KAR5417 appeared rapidly in plasma, an observation consistent with rapid absorption of KAR5585 and efficient conversion to KAR5417.

The mean extent of systemic exposure to the active TPH1 inhibitor, KAR5417, as measured by mean AUC₀₋₂₄ and AUC from time 0 extrapolated to infinity (AUC_(0-inf)), appeared to increase in a dose-proportional manner between the 100 and 700 mg dose levels, with a 6.3-fold and 6.8-fold increase in mean AUC₀₋₂₄ and mean AUC_(0-inf) estimates, respectively, for a 7-fold increase in dose between the 100 mg dose and the 700 mg dose. At doses greater than 700 mg, there were less than dose-proportional increases in AUC₀₋₂₄ and AUC_(0-inf) values, with a 1.7-fold and 1.81-fold increase over the 2.86-fold increase in dose between 700 mg and 2000 mg. The mean apparent terminal half-life (t_(1/2)) after oral administration of KAR5417 increased with increasing dose levels studied, and ranged between 4.7 hours (100 mg dose) and 22.6 hours (2000 mg dose) after oral administration of KAR5585.

Administration of KAR5585 under fed conditions increased the extent and peak of exposure of both KAR5585 and KAR5417, with KAR5417 mean AUC from time 0 extrapolated to infinity (AUC_(0-inf)) increasing 1.8-fold from 3650 ng·hr/mL to 6710 ng·hr/mL and C_(max) increasing 1.8-fold from 485 ng/mL to 860 ng/mL, under fasting and fed conditions, respectively. This was considered to be a clinically relevant change in exposure.

Part 2 MAD PK

KAR5585 was rapidly absorbed following oral administration.

Overall, the mean extent of systemic exposure to KAR5417, as measured by mean AUC from time 0 to 12 hours after dosing (AUC₀₋₁₂) and mean AUC₀₋₂₄, appeared to be comparable under fasted conditions (Part 2, Cohort 1; Day 1 AUC₀₋₁₂=741 hr*ng/mL; Day 14 AUC₀₋₂₄=1650 hr*ng/mL) relative to fed conditions (Part 2, Cohort 2; Day 1 AUC₀₋₁₂=470 hr*ng/mL; Day 14 AUC₀₋₂₄=1220 hr*ng/mL). This observation is in contrast to the food-effect comparison in Part 1, Cohort 3A and 3B KAR5585 400 mg).

In Part 1, a high-fat, high-calorie meal resulted in AUC_(0-inf) of KAR5417 that was approximately 1.8-fold higher following drug administration of KAR5585 400 mg under fed conditions (AUC_(0-inf)=6710 ng·hr/mL) relative to fasting conditions (AUC_(0-inf)=3650 ng·hr/mL).

Steady-state plasma levels of KAR5417 were achieved by Day 7 as assessed by comparison of Day 7 and Day 14 AUC₀₋₁₂ and concentration obtained 12 hours after an administered dose (C_(12 hr)) values following BID oral administration of KAR5585 for 14 days. Accumulation ratios for KAR5417 compared accumulation ratio for C_(max) (R_(Cmax)) and accumulation ratio for AUC₀₋₁₂ (RAUC0-12) for Day7/Day 1 and Day 14/Day 1, and verified that steady-state was achieved by Day 7. The R_(AUC0-12) values for KAR5417 ranged from 2.19-2.57, 1.09-1.34 and 1.65-2.38 for the 100 mg, 200 mg, and 400 mg doses of KAR5585, respectively, suggesting that the PK of KAR5417 was independent of time, and that steady-state was achieved on Day 7 following repeated BID administration for14-days.

Biomarker Results

Dose- and time-dependent reductions were observed in 5-HIAA (a PD marker of 5-HT synthesis) in both plasma and urine. At the highest KAR5585 dose, 400 mg, mean percent change in plasma 5-HIAA concentration was 53.33 from Day 1 to Day 14, whereas mean percent change in subjects randomized to placebo was +20.12. Mean differences of both absolute and percent changes from Day 1 to Day 14 in plasma 5-HIAA concentration were statistically significant in favor of KAR5585 compared to placebo in each dose group and for all doses combined at Day 14.

There was a strong association between urine 5-HIAA/24 hours and KAR5585 dose. At the highest KAR5585 dose, 400 mg, mean percent change in measured urine 5-HIAA was

50.85 from Day 1 to Day 14, whereas mean percent change in subjects randomized to placebo was +3.97. Mean differences of absolute and relative changes from Day 1 to day 14 were statistically significant in favor of KAR5585 in all dose groups except one (the relative change on Day 14 for the KAR5585 100 mg fasting dose group). Results were comparable in urine 5-HIAA/24 hours as adjusted, estimated, and per gram creatinine.

A strong relationship was seen between KAR5417 exposure and 5-HIAA reductions. A strong relationship between both plasma and urine 5-HIAA is supported by the observation that higher exposure to KAR5417, as measured by the PK parameters AUC₀₋₂₄, C_(max), C₁₂, and AUC₀₋₁₂, was associated with greater reduction in 5-HIAA.

Safety Results Part 1, SAD, Period 1—Fasting Administration

KAR5585, administered as a single ascending oral dose (100 mg, 200 mg, 400 mg, 700 mg, 1200 mg or 2000 mg), was safe and well-tolerated in 54 healthy adult subjects.

Part 1, SAD, Period 2—Fed Administration (Food Effect):

KAR5585 400 mg, administered as a single oral dose under fed conditions, was well-tolerated in 9 healthy adult subjects.

Part 2, MAD—Fasting and Fed Administration:

KAR5585, administered as multiple ascending oral doses of 100 mg (fasting), 100 mg (fed), 200 mg (fed), and 400 mg (fed), BID for up to 14 days (27 doses), was safe and well-tolerated in 36 healthy adult subjects. One or more of the MAD Cohorts could have been dosed QD, but none was.

Conclusion

No safety, tolerability, PK, or cardiac-conduction concerns were identified during the course of the study. The administration of KAR5585 was well tolerated throughout the trial: in Part 1 (SAD) Period 1, at doses of 100 mg, 200 mg, 400 mg, 700 mg, 1200 mg or 2000 mg under fasting conditions; in Part 1 (SAD) Period 2, at a dose of 400 mg under fed conditions; and in Part 2 (MAD) at doses of 100 mg (fasting), 100 mg (fed), 200 mg (fed), and 400 mg (fed).

Following administration of KAR5585 (prodrug), KAR5417 (active TPH1 inhibitor) appeared rapidly in plasma, an observation consistent with rapid absorption of KAR5585 and efficient conversion to KAR5417. A strong relationship was seen between KAR5417 exposure and 5-HIAA reductions.

Neither the prodrug (KAR5585) nor the active drug (KAR5417) showed any tendency to increase QTcF in a dose-dependent manner.

As described in the PK SAP, the following PK parameters (as appropriate) were to be generated from KAR5585 and KAR5417 individual plasma concentrations from Part 1, Day 1; and Part 2, Days 1, 7, and 14 within the PK Evaluable Population:

-   -   C_(max1) Maximum concentration, obtained after first daily dose     -   C_(max2) Maximum concentration, obtained after second daily dose     -   C_(12 hr) Concentration, obtained 12 hours after an administered         dose     -   C_(max1)/C_(12 hr) Ratio of maximum concentration, obtained         after first daily dose to the concentration, obtained 12 hours         after first administered daily dose     -   C_(max1)/D Dose-normalized C_(max), obtained after first daily         dose     -   t_(max1) Time to maximum concentration, obtained after first         daily dose     -   t_(max2) Time to maximum concentration, obtained after second         daily dose     -   AUC_(0-tau) Area under the concentration versus time curve from         time 0 to the last quantifiable point within the dosing         interval, using the trapezoidal rule     -   AUC₀₋₁₂ Area under the concentration versus time curve from time         0 to 12 hours after dosing, using the trapezoidal rule     -   AUC₀₋₂₄ Area under the concentration versus time curve from time         0 to 24 hours after dosing, using the trapezoidal rule     -   AUC₀₋₂₄/D Dose-normalized AUC₀₋₂₄ (Day for SAD, Day 1 and Day 7         for MAD)     -   λ_(z) Apparent terminal rate constant after oral administration     -   t_(1/2) Apparent terminal half-life after oral administration     -   AUC_(0-inf) Area under the concentration versus time curve from         time 0 to infinity (SAD only)     -   AUC Extrap Percent of AUC_(0-∞) extrapolated after the last         quantifiable concentration     -   CL/F Apparent oral clearance     -   CL_(ss)/F Apparent oral clearance at steady-state     -   V_(z)/F Apparent oral volume of distribution dependent during         the terminal phase         The doses administered were the following:     -   In Part 1 (SAD) Period 1: single KAR5585 doses of 100 mg, 200         mg, 400 mg, 700 mg, 1200 mg, or 2000 mg or matching placebo in         Cohorts 1 through 6, respectively.     -   In Part 1 (SAD) Period 2: 400 mg in Cohort 3 only.     -   In Part 2 (MAD): KAR5585 100 mg (fasting), 100 mg (fed), 200 mg         (fed), and 400 mg (fed) or matching placebo administered BID         (approximately every 12 hours) for 27 doses in Cohorts 1 through         4, respectively.

Doses were administered using the capsule strengths or sizes shown in Table 9.

TABLE 6 Doses and Capsule Strengths Administered Part 1 (SAD) Part 2 (MAD) Dose Capsules Administered Dose Capsules Administered Cohort (mg) (KAR5585 or Placebo) (mg) (KAR5585 or Placebo) 1 100 Two 50 mg or matching placebo 100 Two 50 mg or matching placebo 2 200 One 200 mg or matching placebo 100 Two 50 mg or matching placebo 3 400 Two 200 mg or matching placebo 200 One 200 mg or matching placebo 4 700 Two 200 mg + one 300 mg or 400 Two 200 mg or matching placebo matching placebo 5 1200 Four 300 mg or matching placebo 6 2000 Six 300 mg or matching placebo + one 200 mg or matching placebo Abbreviations: MAD, multiple ascending dose; SAD, single ascending dose

Pharmacokinetics Results and Tabulations of Individual Subject Data: Plasma Concentrations:

The locations of individual and mean estimates of plasma KAR5585 and KAR5417 concentrations are tabulated below:

Mean plasma concentration-time profiles of KAR5585 and KAR5417 following administration of 100-2000mg KAR5585 under fasting conditions in healthy adult subjects—Cohorts 1-6 (linear and semi-log scales) are presented in FIGS. 3 a and 3 b 5 and FIGS. 4 a and 4 b , respectively. Mean plasma concentration time profiles of KAR5585 and KAR5417 following administration of 400 mg of KAR5417 under fasting and fed conditions—Cohorts 3A and 3B (linear and semi-log scales) are presented in 18 a and 18 b and FIGS. 19 a and 19 b , respectively.

The mean peak plasma KAR5585 concentrations were reached between 1.5 and 3 hours following single-dose fasting oral administration of 100, 200, 400, 700, 1200 and 2000 mg KAR5585. For doses of 100 to 1200 mg KAR5585 the time to peak plasma concentrations of KAR5585 were reached between 0.75 and 6 hours. For the 2000 mg dose the time to peak plasma concentrations of KAR5585 ranged from 0.75 to 12 hours. Mean KAR5585 concentrations increased with increasing doses. KAR5585 concentrations remained above the LLOQ (4.94 ng/mL) up to 4 hours in most subjects following the 100 mg dose, in one subject KAR5585 was BLQ at all time-points and in another subject KAR5585 was measurable up to 8 hours. KAR5585 concentrations remained above the LLOQ (4.94 ng/mL) up to 12 hours following the 400, 700, 1200 and 200 mg doses of KAR5585.

The mean peak plasma KAR5417 concentrations were reached between 1.5 and 3 hours following single-dose fasting oral administration of 100, 200, 400, 700, 1200 and 2000 mg KAR5585. For doses of 100 to 1200 mg KAR5585 the time to peak plasma concentrations of KAR5417 were reached between 2 and 6 hours. For the 2000 mg dose of KAR5585 the time to peak plasma concentrations of KAR5417 ranged from 3 to 12 hours. Mean KAR5417 concentrations increased with increasing doses. KAR5417 concentrations remained above the LLOQ (7.65 ng/mL) up to 12, 24, 36, 48, 48 and 72 hours following the 100, 200, 400, 700, 1200 and 2000 mg doses, respectively. The plasma concentrations of KAR5417 were approximately 10-fold higher than the plasma concentrations of KAR5585.

Following administration of 400 mg KAR5585, peak mean plasma KAR5585 concentrations were observed at later times and sustained longer under fed relative to fasting conditions (t_(max) was 3.0 and 2.07 hour postdose, for fed and fasted states, respectively). The peak mean concentrations were approximately 320% higher following drug administration under fedconditions (C_(max)=169 ng/mL) relative to fasting conditions (C_(max)=52.8 ng/mL). KAR5585 mean concentrations following drug administration under fed conditions were higher relative to fasting conditions and KAR5585 levels were measurable to 24 hours in the fed state, relative to 12 hours in the fasted state.

Following administration of 400 mg KAR5585, peak mean plasma KAR5417 concentrations were observed at similar times under fasting and fed conditions (4 hour postdose). The peak mean concentrations were approximately 177% higher following drug administration underfed conditions (C_(max)=860 ng/mL) relative to fasting conditions (C_(max)=485 ng/mL). KAR5417 mean concentrations following drug administration under fed conditions were higher relative to fasting conditions and KAR5585 levels were measurable to 72 hours in the fed state, relative to 36 hours in the fasted state.

TABLE 7 (Summary of the Mean Pharmacokinetic Parameters of KAR5585 Following a Single Oral Dose of KAR5585 (Cohorts 1-6) (Pharmacokinetic Evaluable Population)) Dose Stat. t_(ma) _(x) ^(a) C_(max) C_(max)/D C₁₂ C_(max)/ AUC_(0-tau) AUC₀₋₁₂ AUC₀₋₂₄ AUC₀₋₂₄/D AUC_(0-∞) V_(z)/F CL/F (mg) Param. t₁ (hr) (hr) (ng/ml) [(ng/mL)/mg] (ng/ml) C₁₂ (hr*ng/ml) (hr*ng/ml) (hr*ng/ml) [(hr*ng/ml)/mg] (hr*ng/ml) (L) (L/hr) 100 N 3 8 8 8 0 0 8 3 3 3 3 3 3 (fasted) Mean 1.89 1.50 13.9 0.139 BLQ ND 35.0 48.1 49.6 0.496 49.5 5680 2210 Cohort 1 SD 1.11 0.750, 6.00 5.35 0.0535 NR ND 24.9 16.2 16.6 0.166 16.5 2830 849 CV % 58.5 NA 38.4 38.4 NR ND 71.1 33.7 33.4 33.4 33.4 49.9 38.5 Geo. Mean 1.71 NA 13.2 0.132 BLQ ND 27.4 46.2 47.5 0.475 47.5 5190 2110 Geo. CV % 58.2 NA 33.6 33.6 NR ND 91.3 36.8 37.9 37.9 37.9 56.8 37.9 200 N 7 8 8 8 0 0 8 7 7 7 7 7 7 (fasted) Mean 3.63 1.78 19.2 0.0961 BLQ ND 61.4 85.8 97.1 0.485 99.5 10300 2220 Cohort 2 SD 2.02 0.750, 3.00 7.84 0.0392 NR ND 27.3 20.5 25.9 0.130 28.0 4060 925 CV % 55.7 NA 40.8 40.8 NR ND 44.5 23.8 26.7 26.7 28.1 39.5 41.6 Geo. Mean 3.17 NA 17.5 0.0875 BLQ ND 52.0 83.3 93.4 0.467 95.3 9610 2100 Geo. CV % 61.6 NA 53.0 53.0 NR ND 85.5 28.0 33.1 33.1 35.4 40.5 35.4 400 N 7 9 9 9 7 7 9 8 7 7 7 7 7 (fasted) Mean 4.08 2.07 52.8 0.132 7.45 8.44 219 246 297 0.742 302 8330 1480 Cohort 3A SD 0.635 1.00, 6.00 34.0 0.0851 0.879 4.64 112 86.6 90.4 0.226 93.2 2240 606 CV % 15.5 NA 64.4 64.4 11.8 55.0 51.0 35.3 30.5 30.5 30.8 26.9 41.1 Geo. Mean 4.04 NA 42.0 0.105 7.40 7.20 176 231 283 0.707 288 8100 1390 Geo. CV % 16.0 NA 88.1 88.1 12.0 72.0 103.0 40.4 36.4 36.4 37.0 25.3 37.0 400 N 9 9 9 9 9 9 9 9 9 9 9 9 9 (fed) Mean 4.45 3.00 169 0.422 21.0 9.35 868 761 885 2.21 915 2780 449 Cohort 3B SD 1.27 1.50, 6.00 36.2 0.0904 7.97 4.39 152 86.7 128 0.321 148 549 85.2 CV % 28.6 NA 21.4 21.4 38.0 47.0 17.5 11.4 14.5 14.5 16.1 19.8 18.9 Geo. Mean 4.26 NA 165 0.413 19.7 8.36 854 756 876 2.19 903 2730 443 Geo. CV % 32.5 NA 22.6 22.6 38.4 55.7 19.6 12.3 15.8 15.8 17.7 21.2 17.7 700 N 8 8 8 8 6 6 8 8 8 8 8 8 8 (fasted) Mean 3.46 1.75 99.4 0.142 10.7 12.2 386 392 432 0.617 436 9120 1810 Cohort 4 SD 0.561 1.00, 3.00 29.3 0.0418 4.73 6.65 135 127 148 0.211 150 4190 701 CV % 16.2 NA 29.4 29.4 44.3 54.4 35.1 32.3 34.2 34.2 34.4 45.9 38.8 Geo. Mean 3.42 NA 95.2 0.136 9.84 10.5 362 372 409 0.584 412 8390 1700 Geo. CV % 16.8 NA 33.7 33.7 47.2 69.7 41.7 36.5 38.0 38.0 38.1 45.0 38.1 1200 N 8 9 9 9 7 7 9 8 8 8 8 8 8 (fasted) Mean 3.58 2.00 83.0 0.0692 11.8 9.78 364 402 453 0.378 462 16900 3850 Cohort 5 SD 1.35 1.00, 4.00 49.0 0.0408 4.33 5.27 189 164 182 0.152 186 12300 4080 CV % 37.8 NA 59.0 59.0 36.8 53.9 51.9 40.7 40.2 40.2 40.2 72.7 106 Geo. Mean 3.40 NA 68.1 0.0568 11.0 8.63 296 356 400 0.333 406 14500 2960 Geo. CV % 33.9 NA 85.0 85.0 44.4 59.3 93.9 67.4 70.9 70.9 73.0 58.7 73.0 2000 N 8 9 9 9 8 8 9 9 9 9 8 8 8 (fasted) Mean 4.84 2.00 104 0.0522 37.4 5.70 773 624 806 0.403 778 25100 3820 Cohort 6 SD 1.05 0.750, 12.0 43.6 0.0218 36.7 3.98 418 239 377 0.188 383 21900 3560 CV % 21.6 NA 41.8 41.8 98.1 69.9 54.0 38.3 46.7 46.7 49.2 87.5 93.1 Geo. Mean 4.76 NA 95.1 0.0475 26.2 4.11 638 558 696 0.348 667 20600 3000 Geo. CV % 20.2 NA 51.6 51.6 105 125 86.9 63.1 73.3 73.3 75.2 63.8 75.2 ^(a)Median and range (Min, Max) presented; Abbreviations: AUC₀₋₁₂, area under the concentration versus time curve from time 0 to 12 hours after dosing, using the trapezoidal rule; AUC₀₋₂₄, area under the concentration versus time curve from time 0 to 24 hours after dosing, using the trapezoidal rule; AUC_(0-tau), area under the concentration-time curve from time 0 to time of last quantifiable concentration; BLQ, below the limit of quantitation; CL/F, apparent oral clearance; CV, coefficient of variation; Geo., geometric; hr, hour; N, number; NA, not applicable; NR, not reported; ND, not determined; Param., parameter; SD, standard deviation; Stat., statistical; t½, apparent terminal half-life after oral administration; t_(max), time of maximum observed concentration; V_(z)/F, volume of distribution during the terminal phase

TABLE 8 (Summary of the Mean Pharmacokinetic Parameters of KAR5417 Following a Single Oral Dose of KAR5585 (Cohorts 1-6) (Pharmacokinetic Evaluable Population)) Dose Stat. t_(1/2) t_(max) ^(a) C_(max) C_(max)/D C₁₂ C_(max)/ AUC_(0-tau) AUC₀₋₁₂ AUC₀₋₂₄ AUC₀₋₂₄/D AUC_(0-∞) (mg) Param. (hr) (hr) (ng/ml) (ng/ml)/mg (ng/ml) C₁₂ (hr*ng/mL) (hr*ng/ml) (hr*ng/ml) (hr*ng/mL)/mg (hr*ng/ml) 100 N 8 9 9 9 8 8 9 8 8 8 8 (fasted) Mean 4.67 4.00 107 1.07 23.8 5.23 718 704 847 8.47 886 Cohort 1 SD 1.35 2.00, 6.00 44.2 0.442 8.61 1.64 358 172 251 2.51 293 CV % 28.9 NA 41.5 41.5 36.1 31.3 49.8 24.5 29.6 29.6 33.1 Geo. Mean 4.51 NA 93.1 0.931 22.5 5.04 605 686 816 8.16 847 Geo. CV % 28.7 NA 72.6 72.6 36.6 28.7 83.2 24.2 29.1 29.1 32.6 200 N 9 9 9 9 9 9 9 9 9 9 9 (fasted) Mean 5.40 3.00 160 0.801 32.9 4.92 1120 947 1160 5.81 1230 Cohort 2 SD 1.08 2.00, 4.00 98.2 0.491 21.5 1.07 816 629 781 3.90 838 CV % 20.0 NA 61.3 61.3 65.5 21.7 72.5 66.4 67.2 67.2 68.0 Geo. Mean 5.29 NA 132 0.658 27.3 4.82 866 771 940 4.70 988 Geo. CV % 21.6 NA 78.8 78.8 72.1 22.4 94.2 79.2 80.8 80.8 83.6 400 N 9 9 9 9 9 9 9 9 9 9 9 (fasted) Mean 11.3 4.00 485 1.21 82.5 6.04 3500 2690 3250 8.12 3650 Cohort 3A SD 9.24 3.00, 6.00 183 0.456 30.5 1.44 1440 1020 1210 3.02 1490 CV % 81.6 NA 37.7 37.7 37.0 23.9 41.0 38.0 37.2 37.2 40.9 Geo. Mean 9.29 NA 444 1.11 75.2 5.90 3120 2460 2970 7.43 3280 Geo. CV % 67.8 NA 52.5 52.5 55.6 22.9 63.4 52.9 53.9 53.9 59.1 400 N 9 9 9 9 9 9 9 9 9 9 9 (fed) Mean 20.5 4.00 860 2.15 169 5.39 6440 4240 5430 13.6 6710 Cohort 3B SD 5.21 3.00, 6.00 224 0.561 34.7 2.06 1000 790 816 2.04 1070 CV % 25.4 NA 26.1 26.1 20.6 38.2 15.6 18.6 15.0 15.0 15.9 Geo. Mean 19.8 NA 831 2.08 165 5.03 6360 4170 5370 13.4 6630 Geo. CV % 33.3 NA 28.8 28.8 20.8 42.2 17.3 20.2 16.2 16.2 17.9 700 N 9 9 9 9 9 9 9 9 9 9 9 (fasted) Mean 13.5 4.00 778 1.11 144 5.97 5840 4350 5310 7.58 6020 Cohort 4 SD 6.04 2.00, 6.00 537 0.767 126 1.53 4350 3010 3800 5.43 4410 CV % 44.9 NA 69.0 69.0 87.4 25.5 74.4 69.3 71.5 71.5 73.2 Geo. Mean 12.1 NA 544 0.777 93.8 5.80 3870 3050 3720 5.31 4170 Geo. CV % 53.6 NA 157 157 153 26.6 180 153 149 149 150 1200 N 9 9 9 9 9 9 9 9 9 9 9 (fasted) Mean 14.9 6.00 896 0.747 213 4.53 7700 5350 6790 5.66 7970 Cohort 5 SD 5.60 3.00, 6.00 453 0.377 130 1.40 4000 2710 3480 2.90 4040 CV % 37.5 NA 50.5 50.5 60.9 30.9 52.0 50.6 51.2 51.2 50.7 Geo. Mean 14.0 NA 764 0.636 175 4.36 6580 4580 5810 4.84 6880 Geo. CV % 41.5 NA 75.0 75.0 81.0 29.0 73.0 73.3 72.8 72.8 69.5 2000 N 9 9 9 9 9 9 9 9 9 9 9 (fasted) Mean 22.6 5.00 1080 0.542 354 4.23 10600 6910 9080 4.54 10900 Cohort 6 SD 5.07 3.00, 12.0 345 0.173 271 1.94 3510 2110 3000 1.50 3570 CV % 22.4 NA 31.9 31.9 76.6 45.8 33.3 30.6 33.0 33.0 32.9 Geo. Mean 22.1 NA 1030 0.515 280 3.67 9880 6550 8520 4.26 10200 Geo. CV % 22.4 NA 37.2 37.2 83.8 70.2 43.9 38.3 42.6 42.6 43.0 ^(a)Median and range (Min, Max) presented; V_(z)/F and CL/F were not calculated for KAR5417 as this is a metabolite of KAR5585 Abbreviations: AUC₀₋₁₂, area under the concentration versus time curve from time 0 to 12 hours after dosing, using the trapezoidal rule; AUC₀₋₂₄, area under the concentration versus time curve from time 0 to 24 hours after dosing, using the trapezoidal rule; AUC_(0-tau), area under the concentration-time curve from time 0 to time of last quantifiable concentration; BLQ, below the limit of quantitation; CL/F, apparent oral clearance; CV, coefficient of variation; Geo., geometric; hr, hour; Max, maximum; Min, minimum; N, number; NA, not applicable; NR, not reported; ND, not determined; Param., parameter; SD, standard deviation; Stat., statistical; t½, apparent terminal half-life after oral administration; tmax, time of maximum observed concentration; V_(z)/F, volume of distribution during the terminal phase.

Pharmacokinetics of KAR5417:

KAR5417 is the active metabolite of the prodrug, KAR5585. Following administration of KAR5585, KAR5417 appeared rapidly in plasma, an observation consistent with rapid absorption of KAR5585 and efficient conversion to KAR5417; all subjects had measurable concentrations of KAR5417 by 0.25 hours postdose, and the median t_(max) was comparable across all fasting Cohorts and ranged between 3 and 6 hours postdose.

In general, mean peak plasma KAR5417 concentrations, as measured by mean C_(max), appeared to increase in a dose-proportional manner from 100-700 mg, mean C_(max) values increased 7.27-fold over the 7-fold increase in dose between the 100 mg dose (Cohort 1) and the 700 mg dose (Cohort 4). At doses greater than 700 mg, there was a moderate increase in C_(max) values, with a 1.38-fold increase over the 2.86-fold increase in dose between 700 mg (Cohort 4) and 2000 mg (Cohort 6).

Overall, the mean extent of systemic exposure to KAR5417, as measured by mean AUC₀₋₂₄ and AUC_(0-inf), appeared to increase in a dose-proportional manner between the 100 and 700 mg dose levels, with a 6.3-fold and 6.8-fold increase in mean AUC₀₋₂₄ and mean AUC_(0-inf) estimates, respectively, for a 7-fold increase in dose between the 100 mg dose (Cohort 1) and the 700 mg dose (Cohort 4). At dose levels greater than 700 mg, there was a less than dose-proportional increase in AUC₀₋₂₄ and AUC_(0-inf) values, with a 1.7-fold and 1.81-fold increase over the 2.86-fold increase in dose between 700 mg (Cohort 4) and 2000 mg (Cohort 6).

Administration of the KAR5585 under fed conditions increased the extent and peak of exposure of KAR5417 with mean AUCs (AUC_(0-inf)) increasing 1.8-fold from 3650 ng·hr/mL to 6710 ng·hr/mL and C_(max) increasing 1.8-fold from 485 ng/mL to 860 ng/mL, under fasting and fed conditions, respectively. This was considered to be a clinically relevant change in exposure.

The mean apparent elimination half-life of KAR5417 increased with increasing dose levels studied, and ranged between 4.67 (100 mg dose) and 22.6 hours (2000 mg dose) after oral administration of KAR5585. The elimination phase of KAR5417 was characterized based on the last 3 to 6 measurable time points.

Dose Proportionality Assessment of KAR5585 and KAR5417

Scatter plots of individual plasma KAR5585 and KAR5417 C_(max) and AUC_(0-inf), versus KAR5585 dose are presented in FIGS. 5 a and 5 b and FIGS. 6 a and 6 b , respectively.

The dose proportionality assessment of plasma KAR5585 and KAR5417 PK parameters are summarized in Table 18. Dose proportionality was assessed for KAR5585 and KAR5417 after single-dose administration using the power model. From plots of exposure (e.g., C_(max), AUC₀₋₁₂, AUC_(0-tau), and/or AUC_(0-inf)) versus dose, increases in exposure were deemed dose proportional if the 90% confidence intervals (CIs) for the slope (β in the following equation(s) included unity (e.g., 1.0).

TABLE 9 (Summary of Dose Proportionality Analysis of Plasma KAR5417 and KAR5585 Pharmacokinetic Parameters AUC₀₋₁₂, AUC_(0-inf), AUC_(0-tau), and C_(max) Following 100 to 2000 mg of KAR5585 Administered as a Single Oral Dose under Fasting Conditions (Pharmacokinetic Evaluable Population)) Degrees 95% Pharmacokinetic of Standard Confidence Parameter Analyte Effect Estimate Freedom Error Interval AUC₀₋₁₂ KAR5417 Intercept 14.5 51 NC NC Slope (β) 0.812 51 0.0898  0.632-0.992 KAR5585 Intercept 1.51 41 NC NC Slope (β) 0.794 41 0.0822  0.629-0.960 AUC_(0-inf) KAR5417 Intercept 12.3 51 NC NC Slope (β) 0.891 51 0.0915 0.707-1.07 KAR5585 Intercept 1.46 39 NC NC Slope (β) 0.822 39 0.0908 0.638-1.01 AUC_(0-tau) KAR5417 Intercept 6.54 52 NC NC Slope (β) 0.975 52 0.101 0.772-1.18 KAR5585 Intercept 0.258 49 NC NC Slope (β) 1.04 49 0.105 0.829-1.25 C_(max) KAR5417 Intercept 1.95 52 NC NC Slope (β) 0.845 52 0.0938 0.657-1.03 KAR5585 Intercept 0.534 49 NC NC Slope (β) 0.707 49 0.0827  0.541-0.873 Dose proportionality was not rejected if the 95% CI for the slope included the value of 1. Abbreviations: AUC₀₋₁₂, area under the concentration versus time curve from time 0 to 12 hours after dosing, using the trapezoidal rule; AUC_(0-inf), area under the concentration versus time curve from time 0 extrapolated to infinity; AUC_(0-tau), area under the concentration-time curve from time 0 to time of last quantifiable concentration; C_(max), maximum observed concentration; CI, confidence interval NC, not calculated.

The 95% CI for the slope of from the power model for AUC_(0-inf) and C_(max) for KAR5417 included the value of 1. Therefore, dose proportionality was concluded for AUC_(0-inf) and C_(max) for KAR5417 in the studied dose range of 100 to 2000 mg KAR5585. Therefore, for KAR5417 AUC_(0-inf) and C_(max) PK parameters, conditions of statistical linearity were met.

The 95% CI for the slope of from the power model for AUC_(0-inf) and C_(max) for KAR5585 did not include the value of 1. Therefore, dose proportionality was not concluded for AUC_(0-inf) and C_(max) for KAR5585 in the studied dose range of 100 to 2000 mg KAR5585. Therefore, for KAR5585 AUC_(0-inf) and C_(max) PK parameters, conditions of statistical linearity were not met and exposure as assessed by AUC_(0-inf) and C_(max) appeared to increase in a less than dose proportional manner.

Food Effect Assessment of KAR5585 and KAR5417:

The statistical results for the food effect assessment following administration of 400 mg KAR5585 under fasting and fed conditions—Cohorts 3A and 3B is presented in Table 10.

TABLE 10 (Summary of Statistical Comparison of Plasma KAR5585 and KAR5417 Pharmacokinetic Parameters AUC_(0-tau), AUC_(0-inf), C_(max): Food Effect Assessment [Cohort 3B (Fed) Versus Cohort 3A (Fasting)] (Pharmacokinetic Evaluable Population)) Geometric Mean Cohort 3B Cohort 3A Geometric LS Parameter (Fed) (Fasting) Mean Ratio 90% CI KAR5585 C_(max) 165 42.0 3.92 2.51-6.13 AUC_(0-tau) 854 176 4.84 3.10-7.56 AUC_(0-inf) 903 288 3.14 2.42-4.07 KAR5417 C_(max) 831 444 1.87 1.47-2.38 AUC_(0-tau) 6360 3120 2.04 1.54-2.69 AUC_(0-inf) 6630 3280 2.02 1.56-2.61 Parameters were In-transformed prior to analysis. Geometric LS means are calculated by exponentiating the LS means from the ANOVA. Geometric Mean Ratio = (test/reference) Cohort 3A (Fasting): 400 mg KAR5585 Administered as a Single Oral Dose Under Fasting Conditions (reference) Cohort 4B (Fed): 400 mg KAR5585 Administered as a Single Oral Dose Under Fed Conditions (test) Abbreviations: ANOVA, analysis of variance; AUC_(0-inf), area under the concentration versus time curve from time 0 extrapolated to infinity; AUC_(0-tau), area under the concentration-time curve from time 0 to time of last quantifiable concentration; CI, confidence interval; C_(max), maximum observed concentration; LS means, least-squares means.

The exposure as assessed by AUC_(0-tau) and AUC_(0-inf), to KAR5585 following administration of 400 mg KAR5585 under fed conditions was approximately 3.1-4.8-fold relative to when the KAR5585 was administered under fasting conditions. Peak exposure (C_(max)) was approximately 3.9-fold higher. The exposure as assessed by AUC_(0-tau) and AUC_(0-inf), to KAR5417 following administration of the 400 mg KAR5585 under fed conditions was approximately 2-fold higher relative to when the KAR5585 was administered under fasting conditions. Peak exposure was approximately 1.87-fold higher. The 90% CI for C_(max), AUC_(0-tau), and AUC_(0-inf) were outside the commonly accepted range of 80-125% suggesting that there was a food effect for AUC_(0-tau) and AUC_(0-inf), and C_(max) for KAR585 and KAR5417. The increase in systemic exposure was considered to be a clinically relevant effect on the oral absorption pharmacokinetics of KAR5585 and KAR5417.

Urine Concentrations:

The locations of individual and mean estimates of urine KAR5585 and KAR5417 concentrations and cumulative excretion tabulated below:

Mean total percent of dose excreted in urine as KAR5417 and mean cumulative urine recoveries in fasted healthy volunteers following administration of 100-2000 mg KAR5585 under fasting conditions in healthy adult subjects—Cohorts 1-6 are presented in FIGS. 7 and 8 .

Mean urine PK parameters for KAR5585 and KAR5417 in healthy volunteers following a single oral dose of KAR5585—Cohorts 1-6 are presented in Table 12 and Table 13, respectively.

In almost all subjects, KAR5585 could not be quantified in urine samples and the amount excreted (Ae) in urine was less than 0.004% of the administered dose at all dose levels (Table 20).

KAR5417 could be quantified in urine samples and the amount excreted (Ae) in urine ranged from 0.102 to 0.331% of the administered dose (Table 13), assuming complete conversion of KAR5585 to KAR5417. The highest amount of KAR5417 excreted, 0.331% of the dose.

FIG. 7 was in the 400 mg fed Cohort (Cohort 3B) and was consistent with the effect of food to increase oral absorption of KAR5585. In general, the amount of KAR5417 excreted in urine was independent of dose in fasted subjects. The majority of urinary excretion of KAR5417 occurred in the first 24 hours after an oral dose of KAR5585.

Renal clearance of KAR5417 was calculated from the area under the plasma time concentration curve (AUC₀₋₉₆) for KAR5417 and the amount of KAR5417 recovered in urine (Table 21). Renal clearance of KAR5417 ranged from 157 to 296 mL/hr and was largely independent of administered dose of KAR5585 (Table 21).

The measurement of KAR5585 and KAR5417 in human urine indicates that renal clearance and elimination of KAR5585 and KAR5417 was a very minor route of elimination of KAR5585 following single dose administration in humans.

TABLE 11 (Mean (±SD) Urine PK Parameters for KAR5585 in Healthy Volunteers Following a Single Oral Dose of KAR5585 (Pharmacokinetic Evaluable Population)) Stat. Amount Total Plasma AUC₀₋₉₆ Dose (mg) Param. Excreted(A_(e)) (μg) Excreted (%) (ng · hr/mL) CL_(R) (mL/hr) 100 N 9 9 3 3 Mean 0 0 49.7 0 SD 0 0 16.6 0 CV % 0 0 33.4 0 200 N 9 9 7 7 Mean 0 0 100 0 SD 0 0 27.7 0 CV % 0 0 27.7 0 400 N 9 9 7 7 (Fasted) Mean 13.8 0.00346 303 51.5 SD 24.6 0.00616 93.2 88.9 CV % 178 178 30.8 173 400 N 9 9 9 9 (Fed) Mean 0 0 917 0 SD 0 0 147 0 CV % 0 0 16.0 0 700 N 9 9 8 9 Mean 0 0 436 0 SD 0 0 150 0 CV % 0 0 34.4 0 1200 N 9 9 8 9 Mean 0 0 463 0 SD 0 0 186 0 CV % 0 0 40.1 0 2000 N 9 9 8 8 Mean 1.50 0.0000751 779 2.30 SD 4.51 0.000225 384 6.50 CV % 300 300 49.3 283 Abbreviations: CV, coefficient of variation; N, number; Param., parameter; SD, standard deviation; Stat., statistical D, standard deviation.

TABLE 12 Mean (±SD) Urine PK Parameters for KAR5417 in Healthy Volunteers Following a Single Oral Dose of KAR5585 (Pharmacokinetic Evaluable Population) Stat. Amount Total Excreted Plasma AUC₀₋₉₆ CL_(R) Dose (mg) Param. Excreted(A_(e)) (μg) (%) (ng · hr/mL) (mL/hr) 100 N 9 9 8 8 Mean 233 0.245 889 296 SD 123 0.129 293 86.1 CV % 52.7 52.7 33.0 29.1 200 N 9 9 9 9 Mean 211 0.111 1240 160 SD 169 0.0885 838 66.8 CV % 80.0 80.0 67.8 41.8 400 N 9 9 9 9 (fasted) Mean 769 0.202 3630 217 SD 376 0.0986 1450 57.2 CV % 48.8 48.8 39.9 26.4 400 N 9 9 9 9 (fed) Mean 1260 0.331 6590 191 SD 259 0.0679 1020 28.9 CV % 20.5 20.5 15.4 15.1 700 N 9 9 9 9 Mean 998 0.150 5990 191 SD 515 0.0773 4360 50.8 CV % 51.6 51.6 72.8 26.5 1200 N 9 9 9 9 Mean 1170 0.102 7910 157 SD 463 0.0405 4010 32.8 CV % 39.6 39.6 50.6 20.9 2000 N 9 9 9 9 Mean 2130 0.112 10700 206 SD 967 0.0508 3480 67.9 CV % 45.5 45.5 32.6 32.9 Abbreviations: CV, coefficient of variation; N, number; PK, pharmacokinetic; Param., parameter; SD, standard deviation; Stat., statistical.

Discussion of Pharmacokinetics Results

KAR5585 is a prodrug for the active TPH1 inhibitor KAR5417. Following KAR5585 administration, KAR5585 was rapidly absorbed the median t_(max) ranged between 1.5 and 3 hours postdose. Following administration of KAR5585, KAR5417 appeared rapidly in plasma, an observation consistent with rapid absorption of KAR5585 and efficient conversion to KAR5417; the median t_(max) was comparable across all fasting Cohorts and ranged between 3 and 6 hours postdose. Following single oral doses of 100 mg to 2000 mg KAR5588 in healthy subjects, the total extent of exposures (AUC_(0-tau), and AUC_(0-inf)) and peak of exposure to KAR5855 and KAR5417 appeared to increase in a proportional manner from 100-700 mg, and in a less than dose proportional manner from 700 mg to 2000 mg.

The mean extent of systemic exposure to the prodrug, KAR5585, as measured by mean AUC_(0-tau) and AUC_(0-inf), appeared to increase in a dose proportional manner between the 100 and 700 mg dose levels, with a 8.7-fold and 8.8-fold increase in mean AUC₀₋₂₄ and mean AUC_(0-inf) estimates, respectively, for a 7-fold increase in dose between the 100 mg dose (Cohort 1) and the 700 mg dose (Cohort 4). At dose greater than 700 mg, there was a less than dose-proportional increase in AUC₀₋₂₄ and AUC_(0-inf) values, with a 1.86-fold and 1.78-fold increase over the 2.86-fold increase in dose between 700 mg (Cohort 4) and 2000 mg (Cohort 6).

The mean extent of systemic exposure to the active TPH1 inhibitor, KAR5417, as measured by mean AUC₀₋₂₄ and AUC_(0-inf), appeared to increase in a dose proportional manner between the 100 and 700 mg dose levels, with a 6.3-fold and 6.8-fold increase in mean AUC₀₋₂₄ and mean AUC_(0-inf) estimates, respectively, for a 7-fold increase in dose between the 100 mg dose (Cohort 1) and the 700 mg dose (Cohort 4). At dose greater than 700 mg, there was a less than dose-proportional increase in AUC₀₋₂₄ and AUC_(0-inf) values, with a 1.7-fold and 1.81-fold increase over the 2.86-fold increase in dose between 700 mg (Cohort 4) and 2000 mg (Cohort 6).

The mean apparent elimination half-life of KAR5585 increased with increasing dose levels studied, and ranged between 1.89 and 4.84 hours after oral administration of KAR5585. The mean apparent elimination half-life of KAR5417 increased with increasing dose levels studied, and ranged between 4.67 (100 mg dose) and 22.6 hours (2000 mg dose) after oral administration of KAR5585.

Mean CL/F estimates of KAR5585 ranged from 1480 to 3850 L/h with the higher CL/F values noted for the 1200 mg (Cohort 5) and 2000 mg (Cohort 6) dose levels and is associated with non-linear PK related to decreased absorption of KAR5585 at the higher dose levels. The V_(z)/F increased with increasing dose levels and ranged between 5680 to 25100 L, the higher V_(z)/F values noted for the 1200 mg (Cohort 5) and 2000 mg (Cohort 6) dose levels was attributed to decreased absorption of KAR5585 at the higher dose levels.

Administration of the KAR5585 under fed conditions increased the extent and peak exposure of KAR5585 with mean AUCs (AUC_(0-inf)) increasing 3-fold from 302 ng·hr/mL to 915 ng·hr/mL and C_(max) increasing 3.2-fold from 52.8 ng/mL to 169 ng/mL, under fasting and fed conditions, respectively. In a similar manner, fed conditions increased the extent and peak of exposure of KAR5417 with mean AUCs (AUC_(0-inf)) increasing 1.8-fold from 3650 ng·hr/mL to 6710 ng·hr/mL and C_(max) increasing 1.8-fold from 485 ng/mL to 860 ng/mL, under fasting and fed conditions, respectively. This was considered to be a clinically relevant change in exposure.

Urinary excretion of KAR5585 and KAR5417 was a minor mechanism of elimination. KAR5585 was not quantifiable in urine in almost all subjects. KAR5417 could be quantified in urine samples and the amount excreted (Ae) in urine ranged from 0.102 to 0.331% of the administered dose of KAR5585.

Pharmacokinetics Conclusions:

KAR5585 is a prodrug for the active TPH1 inhibitor KAR5417. Following KAR5585 administration, KAR5585 was rapidly absorbed the median t_(max) ranged between 1.5 and 3 hours postdose. Following administration of KAR5585, KAR5417 appeared rapidly in plasma, an observation consistent with rapid absorption of KAR5585 and efficient conversion to KAR5417.

The mean extent of systemic exposure to the active TPH1 inhibitor, KAR5417, as measured by mean AUC₀₋₂₄ and AUC_(0-inf), appeared to increase in a dose proportional manner between the 100 and 700 mg dose levels, with a 6.3-fold and 6.8-fold increase in mean AUC₀₋₂₄ and mean AUC_(0-inf) estimates, respectively, for a 7-fold increase in dose between the 100 mg dose and the 700 mg dose. At dose greater than 700 mg, there was a slightly less than dose-proportional increase in AUC₀₋₂₄ and AUC_(0-inf) values, with a 1.7-fold and 1.81-fold increase over the 2.86-fold increase in dose between 700 mg and 2000 mg. The mean apparent elimination half-life of KAR5417 increased with increasing dose levels studied, and ranged between 4.7 hours (100 mg dose) and 22.6 hours (2000 mg dose) after oral administration of KAR5585.

Administration of the KAR5585 under fed conditions, increased the extent and peak of exposure of both KAR5585 and KAR5417, with KAR5417 mean AUCs (AUC_(0-inf)) increasing 1.8-fold from 3650 ng·hr/mL to 6710 ng·hr/mL and C_(max) increasing 1.8-fold from 485 ng/mL to 860 ng/mL, under fasting and fed conditions, respectively. This was considered to be a clinically relevant change in exposure.

PART 2: MULTIPLE ASCENDING DOSE Plasma Concentrations:

Mean plasma concentration-time profiles of KAR5585 and KAR5417 following administration of 100 mg KAR5585 under fasting conditions (Part 2, Cohort 1) and 100, 200 and 400 mg KAR5585 under fed conditions (Part 2, Cohorts 2-4) in healthy adult subjects twice daily for 14-days (semi-log scale) are presented in FIGS. 9 a and 9 b .

Mean (±SD) plasma concentration-time profiles of KAR5585 and KAR5417 following administration of 100 mg KAR5585 under fasting conditions (Part 2, Cohort 1) and 100, 200 and 400 mg KAR5585 under fed conditions (Part 2, Cohorts 2-4) twice-daily for 14-days in healthy adult subjects (semi-log scale) are presented in FIGS. 10 to 13 , respectively.

For Part 2, Cohort 1 (fasted state), samples for KAR5585 and KAR5417 PK analysis were collected on Day 1 and Day 14 following the morning dose of KAR5585. For Part 2, Cohorts 2-4 (fed state), samples for PK analysis were collected on Day 1 and Day 7 following the morning and evening doses and following the single morning dose of KAR5585 that was administered on Day 14.

KAR5585 Plasma Concentrations:

In general, KAR5585 was detectable in circulation at each dose level, and concentrations generally increased with escalating dose. In addition, the length of time with measurable KAR5585 concentrations increased at higher dose levels.

The peak plasma KAR5585 concentrations were reached between 0.75-2.0 and 0.75 and 3.0 hours on Day 1 and Day 14 following oral twice-daily administration of 100 mg of KAR5585, Part 2, Cohort 1 (fasted state). The peak mean concentrations of KAR5585 were 16.7 and 21.3 ng/mL on Day 1 and Day 14, respectively.

For doses of 100, 200 and 400 mg of KAR5585, Part 2, Cohorts 2-4 (fed state), the time to median peak plasma concentrations of KAR5585 ranged from 0.75-4 hours, 1.5 to 6 hours, and 1.5 to 6 hours, respectively, on all dosing days and times. There was some evidence for delayed absorption of KAR5585 on Day 7 for the evening dose, where the t_(max) of KAR5585 were 4 hours, 6 hours, and 6 hours, for the 100, 200 and 400 mg doses, respectively. The Day 7 PM t_(max) values were on the upper end of the t_(max) values determined in Day 1, Day 7 and Day 14.

The peak mean concentrations of KAR5585 for doses of 100, 200 and 400 mg KAR5585, Part 2, Cohorts 2-4 (fed state), increased with dose. For doses of 100, 200 and 400 mg KAR5585, Part 2, Cohorts 2-4 (fed state), the mean C_(max) plasma concentrations of KAR5585 ranged from 18.4-44.1 ng/mL, 42.6-90.9 ng/mL, and 69.0-124 ng/mL, respectively, on all dosing days.

There was some evidence for delayed absorption of KAR5585 on Day 7 for the evening dose, where the mean C_(max) concentrations of KAR5585 for the 100, 200 and 400 mg doses were 28.4 ng/mL, 42.6 ng/mL, and 69 ng/mL, respectively. The Day 7 PM C_(max) values were lower than Day 7 AM C_(max), and at the lower end of the C_(max) values determined for all doses on Day 1, Day 7 and Day 14.

Following administration of 100 mg KAR5585, peak mean plasma KAR5585 concentrations were observed at similar times under fed relative to fasting conditions. Median t_(max) values were 0.75 and 1.5 hour postdose, for fed and fasted states on Day 1, and were 1.8 and 1.0 hour postdose, for fed and fasted states on Day 14, respectively.

The peak mean concentrations of KAR5585 were comparable under fed conditions (Day 1 C_(max)=18.4 ng/mL; Day 14 C_(max)=30 ng/mL,) relative to fasting conditions (Day 1 C_(max)=16.7 ng/mL; Day 14 C_(max)=21.3 ng/mL). This observation is in contrast to the food effect comparison in Part 1, Cohort 3A and 3B (400 mg KAR5585). In Part 1, a high-fat, high calorie meal resulted in peak mean peak concentrations of KAR5585 that were approximately 320% higher following drug administration under fed conditions (C_(max)=169 ng/mL) relative to fasting conditions (C_(max)=52.8 ng/mL). It should be noted that in Part 1, the food effect was evaluated in a cross-over fashion within the same subjects while the food effect was assessed in parallel (different subjects fed versus fasted) in Part 2.

KAR5417 Plasma Concentrations:

In general, circulating concentrations of KAR5417 were notably higher than those for KAR5585 on all days and at all dose levels. In addition, KAR5417 concentrations generally increased with increasing doses of KAR5585.

The peak plasma KAR5417 concentrations were reached between 2.0-6.0 and 2.0 and 4.0 hours on Day 1 and Day 14 following oral twice-daily administration of 100 mg of KAR5585, Part 2, Cohort 1 (fasted state). The mean peak concentrations of KAR5417 were 121 and 194 ng/mL on Day 1 and Day 14, respectively. These values were approximately 7.25-fold and 9.1-fold greater than the corresponding peak mean concentrations of KAR5585.

For doses of 100, 200 and 400 mg of KAR5585, Part 2, Cohorts 2-4 (fed state), the time to median peak plasma concentrations of KAR5417 ranged from 2 to 6 hours, 3 to 8 hours, and 3 to 6 hours, respectively, on all dosing days and times. There was some evidence for a delayed t_(max) for KAR5417 on Day 7 for the evening dose, where the median time to peak plasma concentrations of KAR5417 was 6 hours, 8 hours, and 6 hours, for the 100, 200 and 400 mg doses, respectively. The Day 7 PM median t_(max) values for KAR5417 were on the upper end of the median t_(max) values determined on Day 1, Day 7 and Day 14.

The peak mean concentrations of KAR5417 for doses of 100, 200 and 400 mg KAR5585, Part 2, Cohorts 2-4 (fed state), increased with dose. For doses of 100, 200 and 400 mg KAR5585,Part 2, Cohorts 2-4 (fed state), the mean C_(max) plasma concentrations of KAR5417 rangedfrom 85.9-168 ng/mL, 222-359 ng/mL, and 595-1000 ng/mL, respectively, on all dosing days. There was some evidence for delayed absorption of KAR5585 and a corresponding lowered KAR5417 C_(max) on Day 7 for the evening dose, where the mean C_(max) concentrations of KAR5417 for the 100, 200 and 400 mg doses were 112 ng/mL, 222 ng/mL, and 608 ng/mL, respectively. The Day 7 PM C_(max) values were lower than Day 7 AM C_(max), and on the lower end of the mean C_(max) values determined for all doses on Day 1, Day 7 and Day 14.

Following administration of 100 mg KAR5585, peak mean plasma KAR5417 concentrations were observed at similar times under fed relative to fasting conditions. Median t_(max) values were 2.0 and 3.0 hour postdose, for fed and fasted states on Day 1, and were 3.0 and 3.0 hour postdose, for fed and fasted states on Day 14 respectively). The peak mean concentrations of KAR5417 were comparable under fed conditions (Day 1 C_(max)=85.9 ng/mL; Day 14 C_(max)=168 ng/mL,) relative to fasting conditions (Day 1 C_(max)=121 ng/mL; Day 14 C_(max)=194 ng/mL). This observation is in contrast to the food effect comparison in Part 1, Cohort 3A and 3B (400 mg KAR5585. In Part 1, a high fat, high calorie meal resulted in peak mean concentrations of KAR5417 that were approximately 177% higher following KAR5585 administration under fed conditions (C_(max) 860 ng/mL) relative to fasting conditions (C_(max)=485 ng/mL). Again, it should be noted that the food effect was evaluated in a cross-over fashion in Part 1 while the food effect was assessed in parallel (different subjects fed versus fasted) in Part 2.

Pharmacokinetics Parameters:

The summary of plasma KAR5585 and KAR5417 PK parameters following a repeat-dose oral administration of 100, 200 and 400 mg KAR5585 administered in healthy adult subjects are presented in 15 and Table 16, respectively. Accumulation ratios for KAR5417 and KAR5585 in healthy volunteers following BID oral administration of KAR5585 for 14 days are presented in Table 17.

Pharmacokinetics of KAR5585:

In general, mean peak plasma KAR5585 concentrations, as measured by mean C_(max), appeared to increase in a dose proportional manner from 100-400 mg (Part 2, Cohort 2-4, fed state). Mean C_(max) values increased 4.4-fold over the 4-fold increase in dose between the 100 mg dose (Cohort 2) and the 400 mg dose (Cohort 4) on Day 1. Mean C_(max) values increased ˜3.0-fold over the 4-fold increase in dose between the 100 mg dose (Part 2, Cohort 2) and the 400 mg dose (Part 2, Cohort 4) on Day 14.

Overall, the mean extent of systemic exposure to KAR5585, as measured by mean AUC₀₁₂ and AUC₀₋₂₄, appeared to increase in a dose proportional manner between the 100 and 400 mg dose levels, with a 5.1-fold and 3.6-fold increase in mean AUC₀₋₁₂ and mean AUC₀₋₂₄ estimates, respectively, for a 4-fold increase in dose between the 100 mg dose (Part 2, Cohort 2) and the 400 mg dose (Part 2, Cohort 4) on Day 1. Mean AUC₀₋₁₂ and mean AUC₀₋₂₄ estimates, increased 3.1-fold and 3.6-fold respectively, for a 4-fold increase in dose between the 100 mg dose (Part 2, Cohort 2) and the 400 mg dose (Part 2, Cohort 4) on Day 14.

Overall, the mean extent of systemic exposure to KAR5585, as measured by mean AUC₀₋₁₂ and AUC₀₋₂₄, appeared to be comparable under fasted conditions (Part 2, Cohort 1; Day 1 AUC₀₋₁₂=62.8 hr*ng/mL; Day 14 AUC₀₋₂₄=99.8 hr*ng/mL,) relative to fed conditions (Part 2, Cohort 2; Day 1 AM AUC₀₋₁₂=55.5 hr*ng/mL; Day 14 AUC₀₋₂₄=122 hr*ng/mL,). This observation was in contrast to the food effect comparison in Part 1, Cohort 3A and 3B (400 mg KAR5585). In Part 1, a high fat, high calorie meal resulted in AUC_(0-inf) of KAR5585 that were approximately 3.0-fold higher following drug administration of KAR5585 400 mg under fed conditions (AUC_(0-inf)=915 ng·hr/mL) relative to fasting conditions (AUC_(0-inf)=302 ng·hr/mL). Again, the food effect was evaluated in a cross-over fashion in Part 1 while the food effect was assessed in parallel (different subjects fed versus fasted) in Part 2.

The mean apparent elimination half-life of KAR5585 increased with increasing dose levels, and the mean values were 2.73, 4.08 and 6.45 hrs on Day 14 after oral administration of repeat dose twice-daily administration of 100, 200 and 400 mg of KAR5585 (Part 2, Cohort 2-4, fed state), respectively. The half-life of KAR5585 was 2.49 hrs on Day 14 after oral administration of repeat dose administration of 100 mg in the fasted state, and was comparable to the half-life in the fed state (2.73 hrs) for the 100 mg dose.

Mean CL/F estimates of KAR5585 were 2100, 830 and 1730 L/h for the 100, 200 and 400 mg (Part 2, Cohort 2-4, fed state) doses on Day 1, respectively. Mean steady-state CL/F estimates of KAR5585, in the fed state, were 880, 1200 and 1140 L/h for the 100, 200 and 400 mg (Part 2, Cohort 2-4, fed state) doses on Day 14, respectively. Ranges (mean±SD) were generally overlapping on both days and across the dose range studied here.

There was a slight trend for increasing V_(z)/F increased with increasing dose levels on Day 1, noting that the ranges (mean±SD) were generally overlapping. Mean V_(z)/F was 4100, 3220 and 6800 L for the 100, 200 and 400 mg (Part 2, Cohort 2-4, fed state) doses, respectively. On Day 14, the V_(z)/F increased with increasing dose levels on Day 14 and were 3520, 7010 and 10300 L for the 100, 200 and 400 mg doses, respectively. The highest mean V_(z)/F values were noted for the 400 mg dose (Cohort 4) and is attributed to better characterization of the elimination half-life of KAR5585 at higher doses.

KAR5585 plasma levels achieved steady-state exposure by Day 7 as assessed by comparison of Day 7 and Day 14 AUC₀₋₁₂ and concentration, obtained 12 hours after an administered dose (C_(12 hr)) values following twice daily administration of KAR5585 for 14-days. Accumulation ratios for KAR5585 comparing accumulation ratio for C_(max) (R_(Cmax)) and accumulation ratio for AUC₀₋₁₂ (R_(AUC0-12)) for Day 7/1 and Day 14/Day 1 are presented in Table 17. The R_(AUC0-12) values for KAR5585 ranged from 2.75-3.32, 0.76-0.93 and 1.6-2.01 for the 100, 200 and 400 mg doses of KAR5585 suggesting that the PK of KAR5417 was independent of time and steady-state was achieved on Day 7 following repeated twice-daily administration for 14-days.

Pharmacokinetics of KAR5417

KAR5417 is the active metabolite of the prodrug, KAR5585. Following administration of KAR5585, KAR5417 appeared in plasma, an observation consistent with rapid absorption of KAR5585 and efficient conversion to KAR5417. In general, mean peak plasma KAR5417 concentrations, as measured by mean C_(max), appeared to increase in a greater that dose proportional manner from 100-400 mg (Part 2, Cohort 2-4, fed state). Mean C_(max) values increased 6.9-fold over the 4-fold increase in dose between the 100 mg dose (Cohort 2) and the 400 mg dose (Cohort 4) on Day 1. Day 14 mean C_(max) values increased in an approximately dose proportional manner with an ˜4.5-fold increase in C_(max) over the 4-fold increase in dose between the 100 mg dose (Part 2, Cohort 2) and the 400 mg dose (Part 2, Cohort 4).

Overall, the mean extent of systemic exposure to KAR5417, as measured by mean AUC₀₋₁₂ and AUC₀₋₂₄, appeared to increase in a greater than dose-proportional manner between the 100 and 400 mg dose levels, with a 7.0-fold and 6.45-fold increase in mean AUC₀₋₁₂ and mean AUC₀₋₂₄ estimates, respectively, for 4-fold increase in dose between the 100 mg dose (Part 2, Cohort 2) and the 400 mg dose (Part 2, Cohort 4) on Day 1. Mean AUC₀₋₁₂ and mean AUC₀₋₂₄ estimates, increased 4.6-fold and 4.45-fold respectively, for a 4-fold increase in dose between the 100 mg dose (Part 1, Cohort 2) and the 400 mg dose (Part 2, Cohort 4) on Day 14.

Overall, the mean extent of systemic exposure to KAR5417, as measured by mean AUC₀₋₁₂ and AUC₀₋₂₄, appeared to comparable under fasted conditions (Part 2, Cohort 1; Day 1 AUC₀₋₁₂=741 hr*ng/mL; Day 14 AUC₀₋₂₄=1650 hr*ng/mL,) relative to fed conditions (Part 2, Cohort 2; Day 1 AUC₀₋₁₂=470 hr*ng/mL; Day 14 AUC₀₋₂₄=1220 hr*ng/mL,). This observation is in contrast to the food effect comparison in Part 1, Cohort 3A and 3B (400 mg KAR5585). In Part 1, a high fat, high calorie meal resulted in AUC_(0-inf) of KAR5417 that was approximately 1.8-fold higher following drug administration of KAR5585 400 mg under fed conditions (AUC_(0-inf)=6710 ng·hr/mL) relative to fasting conditions (AUC_(0-inf)=3650 ng·hr/mL). As noted above, the food effect was evaluated in a cross-over fashion in Part 1 while the food effect was assessed in parallel (different subjects fed versus fasted) in Part 2.

The mean apparent elimination half-life of KAR5417 increased with increasing dose levels studied, and mean values were 19.6, 25.0 and 30.6 hrs on Day 14 after oral administration of repeat dose twice-daily administration of 100, 200 and 400 mg of KAR5585 (Part 2, Cohort 2-4, fed state), respectively, determined from a single AM dose on Day 14. The half-life of KAR5417 was 18.8 hrs on Day 14 after oral administration of repeat dose twice-daily administration of 100 mg in the fasted state, and was comparable to the half-life in the fed state (19.6 hrs) for the 100 mg dose. The elimination half —life (30.6 hrs) of KAR5417 on repeat dose administration of 400 mg KAR5585, was similar to the elimination half-life of KAR5417 (20.5 hrs) following single dose administration of 400 mg KAR5585 (Part 1 Cohort 3B).

KAR5417 plasma levels achieved steady-state exposure by Day 7 as assessed by comparison of Day 7 and Day 14 AUC₀₋₁₂ and C_(12 hr) values following twice daily administration of KAR5585 for 14-days. Accumulation ratios for KAR5417 comparing R_(Cmax) and R_(AUC0-12) for Day 7/Day 1 and Day 14/Day 1 are presented in Table 17. The R_(AUC0-12) values for KAR5417 ranged from 2.19-2.57, 1.09-1.34 and 1.65-2.38 for the 100, 200 and 400 mg doses of KAR5585 suggesting that the PK of KAR5417 was independent of time and steady-state was achieved on Day 7 following repeated twice-daily administration for 14-days.

TABLE 13 Mean PK Parameters for KAR5585 in Healthy Volunteers Following BID Oral Administration of KAR5585 for 14 Days (Pharmacokinetic Evaluable Population) Dose C_(max)/D AUC₀₋₂₄/D (mg/ Day/ Stat. t_(1/2) t_(max) ^(a) C_(max) [(ng/ C₁₂ C₂₄ C_(max)/ AUC^(b) ₀₋₁₂ AUC₀₋₂₄ [(hr*ng/ AUC_(0-∞) CL/F dose) Period Param. (hr) (hr) (ng/ml) mL)/mg] (ng/ml) (ng/ml) C₁₂ (hr*ng/ml) (hr*ng/mL) mL)/mg] (hr*ng/ml) V_(z)/F (L) (L/hr) 100/ 1/ N 4 7 7 7 0 0 4 4 4 4 Fasted AM^(c) Mean 2.23 1.5 16.7 0.167 BLQ ND 62.8 71.1 4900 2380 Cohort 1 SD 2.07 0.75, 2.0 5.87 0.0587 NR ND 45.6 60.8 1980 1820 CV % 92.8 NA 35.0 35.0 NR ND 72.7 85.5 40.4 76.3 Geo. Mean 1.73 NA 15.8 0.158 BLQ ND 50.8 54.1 4620 1850 Geo. CV % 88.0 NA 40.2 40.2 NR ND 89.7 105 40.7 105 14 N 7 9 9 9 1 0 1 8 8 8 7 7 8 Mean 2.49 1.0 21.3 0.213 NR BLQ 4.61 92.4 99.8 0.998 105 4420 1450 SD 0.900 0.75, 3.0 7.98 0.0798 NR NR NR 64.5 76.3 0.763 82.7 1370 713 CV % 36.2 NA 37.5 37.5 NR NR NR 69.9 76.4 76.4 78.6 30.9 49.0 Geo. Mean 2.36 NA 20.1 0.201 NR BLQ 4.61 78.2 82.3 0.823 85 4230 1280 Geo. CV % 35.8 NA 34.8 34.8 NR NR NR 63.6 68.9 68.9 75.8 34.5 63.6 100/ 1/ N 4 9 9 9 0 0 4 4 4 4 Fed AM Mean 1.42 0.75 18.4 0.184 BLQ ND 55.5 55.9 4100 2100 Cohort 2 SD 0.455 0.75, 3.0 6.72 0.0672 NR ND 24.9 24.9 1510 945 CV % 32.1 NA 36.4 36.4 NR ND 44.9 44.6 36.7 45.1 Geo. Mean 1.37 NA 17.7 0.177 BLQ ND 51.2 51.7 3830 1940 Geo. CV % 29.3 NA 29.7 29.7 NR ND 49.2 49.2 49.2 49.2 1/ N 7 9 9 9 1 1 8 3 3 7 7 7 PM^(c) Mean 1.87 2.0 44.1 0.441 NR 1.80 143 226 1.13 156 1820 675 SD 0.422 1.5, 8.0 20.4 0.204 NR NR 48.8 90.2 0.451 42.9 588 154 CV % 22.6 NA 46.2 46.2 NR NR 34.0 40.0 40.0 27.4 32.3 22.9 Geo. Mean 1.82 NA 39.8 0.398 NR 1.80 136 214 1.07 152 1730 658 Geo. CV % 25.4 NA 53.9 53.9 NR NR 35.9 41.3 41.3 25.4 35.7 25.4 Dose C_(max)/D AUC₀₋₂₄/D (mg/ Day/ Stat. t_(1/2) t_(max) ^(a) C_(max) [(ng/ C₁₂ C₂₄ C_(max)/ AUC^(b) ₀₋₁₂ AUC₀₋₂₄ [(hr*ng/ AUC_(0-∞) CL/F dose) Period Param. (hr) (hr) (ng/ml) mL)/mg] (ng/ml) (ng/ml) C₁₂ (hr*ng/ml) (hr*ng/mL) mL)/mg] (hr*ng/ml) V_(z)/F (L) (L/hr) 7/ N 8 9 9 9 0 0 8 8 8 8 AM Mean 2.86 2.0 31.3 0.313 BLQ ND 129 139 3360 800 SD 0.544 1.5, 4.0 11.3 0.113 NR ND 25.2 23.4 1110 142 CV % 19.0 NA 35.9 35.9 NR ND 19.5 16.9 32.9 17.7 Geo Mean 2.82 NA 29.8 0.298 BLQ ND 127 137 3210 788 Geo. CV % 17.8 NA 34.4 34.4 NR ND 18.7 16.4 34.1 18.7 7/ N 5 9 9 9 5 5 7 6 6 5 5 5 PM Mean 3.80 4.0 28.4 0.284 6.29 5.37 145 282 1.41 173 3910 717 SD 1.62 1.5, 6.0 15.1 0.151 0.633 3.59 37.6 55.0 0.275 48.5 1850 165 CV % 42.6 NA 53.0 53.0 10.0 66.9 25.9 19.5 19.5 28.0 47.2 23.0 Geo Mean 3.52 NA 25.8 0.258 6.27 4.66 142 278 1.39 168 3550 699 Geo. CV % 46.2 NA 47.2 47.2 10.0 61.2 22.8 18.5 18.5 28.6 53.7 27.1 14 N 7 8 8 8 0 0 0 7 7 7 7 7 7 Mean 2.73 1.8 30.0 0.300 BLQ BLQ ND 115 122 1.22 122 3520 880 SD 0.759 0.75, 3.0 10.0 0.100 NR NR ND 16.0 15.5 0.155 14.5 1290 115 CV % 27.8 NA 33.5 33.5 NR NR ND 13.9 12.7 12.7 11.9 36.7 13.0 Geo Mean 2.64 NA 28.5 0.285 BLQ BLQ ND 114 121 1.21 121 3330 874 Geo. CV % 28.1 NA 35.3 35.3 NR NR ND 13.5 12.3 12.3 11.7 37.7 13.5 200/ 1/ N 9 9 9 9 6 6 9 9 9 9 Fed AM^(c) Mean 2.91 1.5 87.8 0.439 5.61 18.7 257 275 3220 830 Cohort 3 SD 0.806 0.75, 4.0 39.8 0.199 0.268 6.66 89.8 98.3 750 350 CV % 27.7 NA 45.3 45.3 4.8 35.6 34.9 35.7 23.3 42.1 Geo Mean 2.81 NA 80.7 0.403 5.60 17.7 243 258 3130 774 Geo. CV % 30.9 NA 45.0 45.0 4.8 37.4 37.8 40.1 26.0 40.1 Dose C_(max)/D AUC₀₋₂₄/D (mg/ Day Stat. t_(1/2) t_(max) ^(a) C_(max) [(ng/ C₁₂ C₂₄ C_(max)/ AUC_(0-b/12) AUC₀₋₂₄ [(hr*ng/ AUC_(0-∞) CL/F dose) Period Param. (hr) (hr) (ng/ml) mL)/mg] (ng/ml) (ng/ml) C₁₂ (hr*ng/ml) (hr*ng/mL) mL)/mg] (hr*ng/ml) V_(z)/F (L) (L/hr) 400/ 1/ N 9 9 9 9 6 6 9 9 9 9 Fasted AM^(c) Mean 3.14 2. 81.8 0.205 7.49 12.8 281 305 6800 1730 Cohort 4 SD 0.913 1.0, 2.0 34.9 0.0872 0.927 4.03 113 127 2760 1200 CV % 29.1 NA 42.6 42.6 12.4 31.5 40.2 41.5 40.5 69.2 Geo. Mean 3.00 NA 74.0 0.185 7.44 12.2 253 272 2360 1470 Geo. CV % 33.7 NA 54.0 54.0 13.0 36.9 57.3 61.2 39.6 61.2 1/ N 7 9 9 9 9 9 9 9 9 7 7 7 PM^(c) Mean 4.33 3.0 124 0.310 17.1 7.56 528 808 1.01 657 4030 662 SD 1.23 2.0, 4.1 56.1 0.140 3.77 3.25 169 364 0.330 182 1400 241 CV % 28.4 NA 45.2 45.2 22.1 43.0 32.0 32.7 32.7 27.7 34.6 36.4 Geo. Mean 4.18 NA 110 0.276 16.6 6.64 501 760 0.950 633 3820 632 Geo. CV % 29.6 NA 61.0 61.0 25.4 67.9 36.1 41.3 41.3 41.3 32.1 32.1 7/ N 9 9 9 9 9 9 9 9 9 9 AM^(c) Mean 4.90 2.0 95.9 0.240 15.5 6.27 483 588 6220 865 SD 1.85 1.5, 4.0 30.7 0.0769 3.86 1.76 102 124 3230 198 CV % 37.7 NA 32.1 32.1 24.8 28.0 21.1 21.1 51.9 22.9 Geo. Mean 4.65 NA 93.3 0.228 15.1 6.04 473 577 5680 846 Geo. CV % 33.6 NA 34.7 34.7 24.6 30.5 22.4 21.9 45.0 22.4 7/ N 4 9 9 9 9 9 9 9 9 4 4 4 PM^(c) Mean 4.93 6.0 69.0 0.172 20.8 3.90 404 887 1.11 538 6950 969 SD 0.354 2.0, 8.0 30.8 0.0769 5.76 2.92 96.3 185 0.231 77.1 1800 196 CV % 7.17 NA 44.6 44.6 27.6 74.7 23.8 20.9 20.9 14.3 25.9 20.3 Geo. Mean 4.92 NA 63.4 0.158 20.1 — 3.15 394 870 1.09 534 6770 954 Geo. CV % 7.22 NA 45.8 45.8 29.9 75.7 23.9 21.6 21.6 14.3 27.5 21.0 Dose C_(max)/D AUC₀₋₂₄/D (mg/ Day/ Stat. t_(1/2) t_(max) ^(a) C_(max) [(ng/ C₁₂ C₂₄ C_(max)/ AUC₀₋₁₂ ^(b) AUC₀₋₂₄ [(hr*ng/ AUC_(0-∞) CL/F dose) Period Param. (hr) (hr) (ng/ml) mL)/mg] (ng/ml) (ng/ml) C₁₂ (hr*ng/ml) (hr*ng/mL) mL)/mg] (hr*ng/ml) V_(z)/F (L) (L/hr) 1/ N 8 9 9 9 9 9 9 9 9 8 8 8 PM^(c) Mean 4.34 3.0 90.9 0.455 9.92 9.98 344 601 1.5 417 3120 507 SD 1.03 1.5, 6.0 29.6 0.148 2.81 4.65 90.8 156 0.389 102 785 129 CV % 23.7 NA 32.6 32.6 28.4 46.5 26.4 25.9 25.9 24.4 25.2 25.6 Geo. Mean 4.24 NA 85.4 0.427 9.57 8.93 333 583 1.46 406 3010 493 Geo. CV % 24.2 NA 41.8 41.8 29.4 56.2 27.5 26.5 26.5 25.5 30.2 25.5 7/ N 9 9 9 9 8 8 9 9 9 9 AM^(c) Mean 4.30 3.0 48.8 0.244 7.71 6.69 230 273 5960 978 SD 0.938 2.0, 4.0 18.9 0.0943 2.78 2.02 83.9 101 2150 342 CV % 21.8 NA 38.7 38.7 36.1 30.2 36.5 36.9 36.0 35.0 Geo. Mean 4.21 NA 45.6 0.228 7.36 6.43 217 256 5610 924 Geo. CV % 22.1 NA 40.7 40.7 31.7 31.3 37.4 39.1 38.4 37.4 7/ N 2 8 8 8 8 8 8 8 8 2 2 2 PM^(c) Mean 6.20 6.0 42.6 0.213 12.2 4.20 207 431 1.08 290 8710 890 SD NR 2.0, 8.0 19.5 0.0976 5.46 3.14 53.9 133 0.332 NR NR NR CV % NR NA 45.8 45.8 44.6 74.7 26.1 30.8 30.8 NR NR NR Geo. Mean 5.58 NA 39.1 0.195 11.3 3.46 201 414 1.03 289 7000 869 Geo. CV % NR NA 46.4 43.6 24.6 68.5 25.7 30.8 30.8 NR NR NR 14 N 7 8 8 8 3 0 3 7 7 7 7 7 7 Mean 4.08 2.0 48.8 0.244 NR BLQ 11.2 188 212 1.06 216 7010 1200 SD 1.06 1.0, 6.0 24.8 0.124 NR NR 4.75 69.9 81.7 0.409 83.9 3070 447 CV % 25.9 NA 50.8 50.8 NR NR 42.3 37.2 38.6 38.6 38.9 43.8 37.2 Geo. Mean 3.94 NA 43.5 0.217 NR BLQ 10.5 177 199 0.993 202 6430 1130 Geo. CV % 31.0 NA 56.0 56.0 NR NR 48.9 39.6 40.9 40.9 41.3 48.2 39.6 Dose C_(max)/D AUC₀₋₂₄/D (mg/ Day/ Stat. t_(1/2) t_(max) ^(a) C_(max) [(ng/ C₁₂ C₂₄ C_(max)/ AUC_(0-b/12) AUC₀₋₂₄ [(hr*ng/ AUC_(0-∞) CL/F dose) Period Param. (hr) (hr) (ng/ml) mL)/mg] (ng/ml) (ng/ml) C₁₂ (hr*ng/ml) (hr*ng/mL) mL)/mg] (hr*ng/ml) V_(z)/F (L) (L/hr) 14 N 9 9 9 9 9 3 9 9 9 9 9 9 9 Mean 6.45 1.5 89.0 0.222 11.4 NR 8.17 366 444 1.11 482 10300 1140 SD 2.48 1.0, 2.0 26.6 0.0664 3.37 NR 3.00 72.6 103 0.257 138 3420 270 CV % 38.4 NA 29.8 29.8 29.6 NR 36.7 19.8 23.2 23.2 28.6 33.4 23.7 Geo. Mean 6.08 NA 85.3 0.213 11.0 NR 7.76 359 433 1.08 465 9780 1110 Geo. CV % 36.1 NA 32.0 32.0 28.4 NR 33.7 21.7 24.4 24.4 29.2 33.6 21.7 ^(a)Median and range (Min, Max) Presented; ^(b)AUC₀₋₄ substituted for AUC₀₋₁₂ when WinNonlin did not calculate the latter and tlast was >11.90 hr; ^(c)AM refers to a morning dose (8AM) and PM refers to an evening dose (8PM) Shaded cells without data are where PK sampling regimens relative to time of dose administration did not support calculations of PK parameters (eg, no C24 hr values for BID dosing in Day 1 and Day 7) Abbreviations: AM, before noon (ante meridiem) AUC₀₋₁₂, area under concentration versus time curve from time 0 to 12 hours after dosing, using the trapezoidal rule; AUC₀₋₂₄, area under the concentration versus time curve from time 0 to 24 hours after dosing, using the trapezoidal rule; AUC_(0-t), area under the concentration-time curve from time 0 to time of last quantifiable concentration; BLQ, below the limit of quantitation; CL/F, apparent oral clearance; NA, not applicable; ND, not determined; NR, not reported; Pararn., parameter; PK, pharmacokinetic; PM, after noon (post meridiem). SD, standard deviation; Stat., statistical; t½, apparent terminal half-life after oral administration; t_(max), time of maximum observed concentration; Vz/F, volume of distribution during the terminal phase

TABLE 14 (Mean PK Parameters for KAR5417 in Healthy Volunteers Following BID Oral Administration of KAR5585 for 14 Days (Pharmacokinetic Evaluable Population)) C_(max)/D C_(max) (ng/ C₁₂ C₂₄ AUC₀₋₂₄ AUC₀₋₂₄/D AUC_(0-∞) Stat. t_(1/2) t_(ma) _(x) ^(a) (ng/ ml)/ (ng/ (ng/ C_(max)/ AUC₀₋₁₂ ^(b) (hr*ng/ (hr*ng/ml)/ (hr*ng Cohort Day Period Param. (hr) (hr) ml) mg ml) ml) C₁₂ (hr*ng/ml) mL) mg /mL) R_(Cma) ^(d) _(x) R_(AUC0) ^(d) ₋₁₂ 100 1 AM^(c) N 7 9 9 9 8 8 8 7 (fasted) Mean 3.43 3.0 121 1.21 21.3 6.08 741 876 Cohort 1 SD 0.751 2.0, 6.0 83.3 0.833 10.6 1.32 491 545 CV % 21.9 NA 68.9 68.9 49.5 21.7 66.3 62.2 Geo. Mean 3.37 NA 101 1.01 19.5 5.93 643 769 Geo. CV % 21.3 NA 70.4 70.4 45.4 24.8 56.8 55.9 14 — N 9 9 9 9 9 9 9 9 9 9 9 9 8 Mean 18.8 3.0 194 1.94 46.3 19.7 4.25 1280 1650 16.5 2220 2.56 1.83 SD 6.67 2.0, 4.0 87.7 0.877 21.0 8.40 0.539 620 782 7.82 1130 2.96 0.940 CV % 35.5 NA 45.2 45.2 45.3 42.5 12.7 48.4 47.3 47.3 50.9 116 51.4 Geo. Mean 17.6 NA 177 1.77 42.0 18.1 4.21 1150 1490 14.9 1960 1.75 1.64 Geo. CV % 42.0 NA 49.0 49.0 51.5 47.5 14.2 53.0 52.1 52.1 59.2 101 52.5 100 1 AM^(c) N 9 9 9 9 9 9 9 9 (fed) Mean 4.18 2.0 85.9 0.859 17.2 5.26 470 564 Cohort 2 SD 0.516 1.5, 4.0 40.4 0.404 9.54 1.24 247 288 CV % 12.3 NA 47.0 47.0 55.4 23.6 52.5 51.0 Geo. Mean 4.16 NA 79.2 0.792 15.4 5.14 428 516 Geo. CV % 12.2 NA 42.6 42.6 49.9 23.3 45.1 44.0 PM^(c) N 7 9 9 9 9 9 9 9 9 7 Mean 7.00 3.0 130 1.3 43.1 3.31 748 1220 6.09 1140 SD 0.837 2.0, 12 59.8 0.598 21.6 1.36 251 454 2.27 394 CV % 12.0 NA 46.0 46.0 50.0 41.0 33.5 37.3 37.3 34.5 Geo. Mean 6.95 NA 119 1.19 39.5 3.01 714 1160 5.78 1090 Geo. CV % 13.1 NA 45.9 45.9 44.0 53.2 32.6 33.8 33.8 35.7 7 AM^(c) N 9 9 9 9 9 9 9 9 9 9 Mean 5.31 3.0 168 1.68 45.1 3.69 1080 1410 2.07 2.57 SD 0.975 2.0, 4.0 60.9 0.609 9.46 0.837 296 300 0.502 0.8 CV % 18.4 NA 36.2 36.2 21.0 22.7 27.4 21.3 24.2 31.2 Geo. Mean 5.24 NA 160 1.60 44.3 3.61 1050 1380 2.02 2.45 Geo. CV % 18.2 NA 33.8 33.8 20.5 21.4 26.9 20.5 24.6 34.0 PM^(c) N 4 9 9 9 9 9 9 9 9 4 9 9 Mean 8.10 6.0 112 1.12 62.3 1.8 902 1990 9.93 1730 1.43 2.19 SD 2.11 2.0, 8.0 46.4 0.464 11.5 0.629 254 533 2.67 337 0.552 0.806 CV % 26.0 NA 41.3 41.3 18.4 34.9 28.2 26.8 26.8 19.5 38.6 36.8 Geo. Mean 7.91 NA 106 1.06 61.4 1.73 875 1930 9.64 1700 1.34 2.04 Geo. CV % 25.6 NA 36.0 36.0 19.0 30.8 25.9 25.8 25.8 19.2 41.8 43.0 14 AM^(c) N 8 8 8 8 8 8 8 8 8 8 8 8 Mean 19.6 2.0 168 1.68 36.4 19.6 4.56 943 1270 1830 1.98 2.23 SD 5.30 1.5, 4.0 67.2 0.672 9.46 4.11 1.43 296 366 455 0.650 0.818 CV % 27.0 NA 40.0 40.0 26.0 20.9 31.5 31.4 28.8 24.9 32.8 36.7 Geo. Mean 19.0 NA 155 1.55 35.4 19.3 4.37 900 1220 1780 1.90 2.09 Geo. CV % 29.0 NA 47.7 47.7 25.7 21.0 31.6 34.4 30.9 25.7 31.1 40.2 200 1 AM^(c) N 9 9 9 9 9 9 9 9 (fed) Mean 4.49 3.0 357 1.79 60.7 6.07 1700 2090 Cohort 3 SD 0.977 2.0, 4.0 142 0.709 25.1 1.36 605 785 CV % 21.8 NA 39.7 39.7 41.4 22.5 35.5 37.5 Geo. Mean 4.41 NA 336 1.68 56.6 5.94 1620 1980 Geo. CV % 20.1 NA 37.4 37.4 41.0 21.9 35.0 36.3 PM^(c) N 6 9 9 9 9 9 9 9 9 6 Mean 6.03 4.0 322 1.61 112 3.24 2000 3700 9.26 2720 SD 1.09 2.0, 6.0 103 0.513 56.0 1.28 680 1260 3.16 775 CV % 18.1 NA 31.9 31.9 50.2 39.4 34.0 34.1 34.1 28.5 Geo. Mean 5.95 NA 307 1.54 102 3.00 1910 3540 8.85 2640 Geo. CV % 18.5 NA 33.8 33.8 43.6 44.6 31.5 31.9 31.9 27.9 7 AM^(c) N 8 9 9 9 9 9 9 8 9 9 Mean 4.61 4.0 359 1.80 93.1 3.83 2300 2800 1.00 1.34 SD 0.643 2.0, 6.0 176 0.879 38.8 0.393 1010 1280 0.176 0.198 CV % 13.9 NA 49.0 49.0 41.7 10.3 44.0 45.8 17.5 14.8 Geo. Mean 4.57 NA 332 1.66 87.2 3.81 2150 2610 0.989 1.33 Geo. CV % 14.3 NA 40.6 40.6 38.6 10.6 38.5 39.4 18.5 14.6 PM^(c) N 2 8 8 8 8 8 8 8 8 2 8 8 Mean 6.64 8.0 222 1.11 145 1.90 1540 3810 9.52 2280 0.711 0.984 SD NR 3.0, 12 71.3 0.356 74.3 1.31 413 1440 3.60 NR 0.376 0.276 CV % NR NA 32.1 32.1 51.1 68.9 26.9 37.8 37.8 NR 52.9 28.0 Geo. Mean 6.48 NA 212 1.06 131 1.62 1490 3620 9.05 2270 0.645 0.955 Geo. CV % NR NA 33.6 33.6 51.3 61.1 25.8 33.5 33.5 NR 46.5 26.1 14 AM^(c) N 8 8 8 8 8 8 8 8 8 8 8 8 Mean 25.0 3.0 319 1.59 63.5 32.6 4.81 1820 2380 3480 0.901 1.09 SD 7.06 2.0, 6.0 175 0.877 24.9 10.7 0.991 887 1090 1430 0.304 0.235 CV % 28.3 NA 55.1 55.1 39.3 33.0 20.6 48.7 45.7 41.1 33.7 21.5 Geo. Mean 24.1 NA 281 1.40 59.6 31.2 4.72 1670 2200 3250 0.854 1.07 Geo. CV % 30.2 NA 57.2 57.2 38.9 32.0 22.5 45.7 43.1 41.1 36.8 24.0 400 1 AM^(c) N 9 9 9 9 9 9 9 9 (fed) Mean 3.80 3.0 595 1.49 99.3 5.97 3280 3780 Cohort 4 SD 0.883 2.0, 4.0 273 0.682 43.6 0.981 1550 1690 CV % 23.2 NA 45.8 45.8 44.0 16.4 47.4 44.8 Geo. Mean 3.71 NA 532 1.33 90.2 5.90 2920 3400 Geo. CV % 24.8 NA 57.8 57.8 51.3 16.9 58.5 54.6 PM^(c) N 5 9 9 9 9 9 9 9 9 5 Mean 5.31 4.0 739 1.85 252 3.05 4590 7870 9.83 7090 SD 0.456 3.0, 8.0 343 0.859 76.7 1.29 1550 2830 3.53 1970 CV % 8.6 NA 46.4 46.4 30.4 42.2 33.7 35.9 35.9 27.8 Geo. Mean 5.3 NA 670 1.68 242 2.77 4340 7430 9.28 6860 Geo. CV % 8.6 NA 50.6 50.6 31.8 52.9 37.1 37.3 37.3 30.0 7 AM^(c) N 6 9 9 9 9 9 9 6 9 9 Mean 3.94 4.0 1000 2.51 240 4.36 6600 7840 2.00 2.38 SD 0.798 3.0, 6.0 315 0.788 103 0.654 2340 2650 0.991 1.17 CV % 20.2 NA 31.4 31.4 43.0 15.0 35.4 33.9 49.5 49.1 Geo. Mean 3.89 NA 960 2.40 222 4.32 6250 7480 1.81 2.15 Geo. CV % 18.1 NA 31.9 31.9 41.6 15.6 35.7 34.6 49.5 51.3 PM^(c) N 2 9 9 9 9 9 9 9 9 2 9 9 Mean 5.10 6.0 608 1.52 396 1.80 4610 11200 14.0 6520 1.18 1.63 SD NR 3.0, 12 201 0.503 198 0.961 1590 3890 4.86 NR 0.498 0.737 CV % NR NA 33.1 33.1 50.0 53.4 34.5 34.7 34.7 NR 42.4 45.2 Geo. Mean 5.08 NA 574 1.44 356 1.61 4360 10600 13.3 6450 1.08 1.50 Geo. CV % NR NA 38.5 38.5 51.3 50.1 36.9 35.8 35.8 NR 46.9 45.4 14 AM^(c) N 9 9 9 9 9 9 9 9 9 9 9 9 9 Mean 30.6 3.0 749 1.87 149 76.6 5.20 4350 5660 14.1 8500 1.54 1.65 SD 3.84 2.0, 3.0 230 0.574 56.8 25.5 1.08 1420 1870 4.68 2820 0.967 1.06 CV % 12.5 NA 30.6 30.6 38.1 33.3 20.8 32.6 33.1 33.1 33.1 63.0 63.9 Geo. Mean 30.4 NA 717 1.79 140 73.3 5.11 4160 5410 13.5 8120 1.35 1.43 Geo. CV % 12.5 NA 32.8 32.8 37.4 31.6 20.5 32.2 32.3 32.3 31.9 54.0 59.6 ^(a)Median and range (Min, Max) presented; ^(b)AUC_(0-tau) substituted for AUC₀₋₁₂ when WinNonlin did not calculate the latter and t_(last) was >11.90 hr; ^(c)AM refers to a morning dose (8AM) and PM refers to an evening dose (8PM) ^(d)R_(Cmax) and R_(AUC0-12) refer to accumulation ratios for Day 7/Day 1 or Day 14/Day 1 PK parameters Abbreviations: AM, before noon (ante meridiem); AUC₀₋₁₂, area under the concentration versus time curve from time 0 to 12 hours after dosing, using the trapezoidal rule; AUC₀₋₂₄, area under the concentration versus time curve from time 0 to 24 hours after dosing, using the trapezoidal rule; AUC_(0-tau), area under the concentration-time curve from time 0 to time of last quantifiable concentration; BID, twice daily; BLQ, below the limit of quantitation; hr, hour; Max, maximum; Min, minimum; NA, not applicable; ND, not determined; NR, not reported. PK, pharmacokinetic; PM, after noon (post meridiem); Param., parameter; R_(AUC0-12), accumulation ratio for AUC₀₋₁₂; R_(Cmax), accumulation ratio for C_(max); SD, standard deviation; Stat., statistical; t½, apparent terminal half-life after oral administration; t_(max), time of maximum observed concentration.

TABLE 15 (Accumulation Ratios for KAR5417 and KAR5585 in Healthy Volunteers Following BID Oral Administration of KAR5585 for 14 Days (Pharmacokinetic Evaluable Population)) Dose Post AM (mg/feeding or PM Stat. KAR5585 KAR5417 status) Day Dose Param. R_(Cmax) R_(AUC0-12) R_(Cmax) R_(AUC0-12) 100/ 14 AM N 7 4 9 8 Fasted Mean 1.30 1.85 2.56 1.83 Cohort 1 SD 0.592 0.870 2.96 0.940 CV % 45.5 47.0 116 51.4 Geo. Mean 1.20 1.70 1.75 1.64 Geo. CV % 43.0 52.1 101 52.5 100/ 7 AM N 9 3 9 9 Fed Mean 1.74 3.32 2.07 2.57 Cohort 2 SD 0.469 1.10 0.502 0.8 CV % 26.9 33.2 24.2 31.2 Geo. Mean 1.69 3.18 2.02 2.45 Geo. CV % 28.7 38.0 24.6 34.0 7 PM N 9 3 9 9 Mean 1.66 3.11 1.43 2.19 SD 1.03 2.18 0.552 0.806 CV % 62.1 70.1 38.6 36.8 Geo. Mean 1.46 2.68 1.34 2.04 Geo. CV % 54.5 71.6 41.8 43.0 14 AM N 8 3 8 8 Mean 1.68 2.75 1.98 2.23 SD 0.465 0.938 0.650 0.818 CV % 27.7 34.1 32.8 36.7 Geo. Mean 1.62 2.62 1.90 2.09 Geo. CV % 30.2 40.8 31.1 40.2 200/ 7 AM N 9 9 9 9 Fed Mean 0.589 0.933 1.00 1.34 Cohort 3 SD 0.175 0.328 0.176 0.198 CV % 29.8 35.2 17.5 14.8 Geo. Mean 0.565 0.892 0.989 1.33 Geo. CV % 31.1 30.6 18.5 14.6 7 PM N 8 8 8 8 Mean 0.597 0.895 0.711 0.984 SD 0.379 0.304 0.376 0.276 CV % 63.4 34.0 52.9 28.0 Geo. Mean 0.515 0.854 0.645 0.955 Geo. CV % 59.7 33.3 46.5 26.1 14 AM N 8 7 8 8 Mean 0.643 0.761 0.901 1.09 SD 0.323 0.230 0.304 0.235 CV % 50.2 30.2 33.7 21.5 Geo. Mean 0.573 0.730 0.854 1.07 Geo. CV % 56.1 32.6 36.8 24.0 400/ 7 AM N 9 9 9 9 Fed Mean 1.32 2.01 2.00 2.38 Cohort 4 SD 0.627 0.872 0.991 1.17 CV % 47.4 43.3 49.5 49.1 Geo. Mean 1.23 1.87 1.81 2.15 Geo. CV % 37.1 40.8 49.5 51.3 7 PM N 9 9 9 9 Mean 0.971 1.71 1.18 1.63 SD 0.444 0.810 0.498 0.737 CV % 45.7 47.3 42.4 45.2 Geo. Mean 0.857 1.56 1.08 1.50 Geo. CV % 63.6 48.1 46.9 45.4 14 AM N 9 9 9 9 Mean 1.28 1.60 1.54 1.65 SD 0.647 0.938 0.967 1.06 CV % 50.4 58.7 63.0 63.9 Geo. Mean 1.15 1.42 1.35 1.43 Geo. CV % 52.5 52.2 54.0 59.6 Abbreviations: AM, before noon (ante meridiem); BID, twice daily; Geo., geometric; hr, hour; N, number; PM, after noon (post meridiem); R_(AUC0-12), accumulation ratio for AUC₀₋₁₂; R_(Cmax), accumulation ratio for C_(max); SD, standard deviation; t½, apparent terminal half-life after oral administration.

Dose Proportionality Assessment of KAR5585 and KAR5417:

Scatter plots of individual plasma KAR5585 and KAR5417 Day 1 and Day 14 C_(max) KAR5585 dose are presented in FIGS. 14 a and 14 b and FIGS. 15 a and 15 b , respectively. Scatter plots of individual plasma KAR5585 and KAR5417 Day 1 and Day 14 AUC₀₋₁₂ KAR5585 dose are presented in FIGS. 16 a and 16 b and FIGS. 17 a and 17 b . The dose proportionality assessment of plasma KAR5585 and KAR5417 PK parameters are summarized in Table 16. Dose proportionality was assessed for KAR5585 and KAR5417 after repeat-dose, twice-daily administration of KAR5585 using the power model. From plots of exposure (i.e., C_(max), AUC₀₋₁₂ and/or AUC_(0-inf)) versus dose, increases in exposure were deemed dose proportional if the 95% confidence intervals (CIs) for the slope (β included unity (e.g., 1.0). The CIs presented in Table 16 indicate that C_(max), AUC₀₋₁₂ and/or AUC_(0-inf) for both KAR5585 and KAR5417 increase in dose proportional manner from 100-400 mg of KAR5585 (Part 2, Cohorts 2-4) in the fed state.

TABLE 16 (Summary of Dose Proportionality Analysis of Plasma KAR5417 and KAR5585 Pharmacokinetic Parameters AUC₀₋₁₂, AUC_(0-inf), and C_(max) Following 100 to 400 mg of KAR5585 Administered as Repeat Oral Doses under Fed Conditions (Pharmacokinetic Evaluable Population)) 95% PK Day Degrees of Standard Confidence Parameter (AM) Analyte Effect Estimate Freedom Error Interval AUC₀₋₁₂ 1 KAR5417 Intercept 0.828 25 NC NC Slope (β) 1.38 25 0.161  1.05-1.71 KAR5585 Intercept 0.969 20 NC NC Slope (β) 0.964 20 0.245 0.453-1.47 7 KAR5417 Intercept 2.63 25 NC NC Slope (β) 1.29 25 0.114  1.05-1.52 KAR5585 Intercept 1.51 24 NC NC Slope (β) 0.952 24 0.0950 0.756-1.15 14 KAR5417 Intercept 5.18 23 NC NC Slope (β) 1.11 23 0.127 0.846-1.37 KAR5585 Intercept 2.37 21 NC NC Slope (β) 0.832 21 0.0951 0.634-1.03 AUC_(0-inf) 1 KAR5417 Intercept 1.12 25 NC NC Slope (β) 1.36 25 0.159  1.03-1.69 KAR5585 Intercept 0.817 20 NC NC Slope (β) 1.01 20 0.255 0.474-1.54 C_(max) 1 KAR5417 Intercept 0.167 25 NC NC Slope (β) 1.37 25 0.169  1.03-1.72 KAR5585 Intercept 0.198 25 NC NC Slope (β) 1.03 25 0.193 0.636-1.43 7 KAR5417 Intercept 0.393 25 NC NC Slope (β) 1.29 25 0.118  1.05-1.54 KAR5585 Intercept 0.688 25 NO NC Slope (β) 0.809 25 0.121 0.561-1.06 14 KAR5417 Intercept 0.877 23 NC NC Slope (β) 1.11 23 0.153 0.794-1.43 KAR5585 Intercept 0.705 23 NC NC Slope (β) 0.794 23 0.139 0.508-1.08 Abbreviations: AM, before noon (ante meridiem); AUC₀₋₁₂, area under the concentration versus time curve from time 0 to 12 hours after dosing, using the trapezoidal rule; AUC₀₋₂₄, area under the concentration versus time curve from time 0 to 24 hours after dosing, using the trapezoidal rule; AUC_(0-inf), area under the concentration versus time curve from time 0 extrapolated to infinity; NC, not calculated; PK, pharmacokinetic.

Discussion of Pharmacokinetics Results:

Following KAR5585 administration, KAR5585 was rapidly absorbed. For doses of 100, 200 and 400 mg KAR5585, Part 2, Cohorts 2-4 (fed state), the median time to peak plasma concentrations of KAR5585 ranged from 0.75-4 hours, 1.5 to 6 hours, and 1.5 to 6 hours, respectively, on all dosing days and times. There was some evidence for delayed absorption of KAR5585 on Day 7 for the evening dose, where the median time to peak plasma concentrations of KAR5585 were 4 hours, 6 hours, and 6 hours, for the 100, 200 and 400 mg doses, respectively. The Day 7 PM t_(max) values were on the upper end of the t_(max) values determined in

Day 1, Day 7 and Day 14. This was not observed on Day 1.

Peak plasma KAR5585 concentrations, as measured by mean C_(max), increased in a dose proportional manner from 100-400 mg (Part 2, Cohort 2-4, fed state). Mean C_(max) values increased 4.4-fold over the 4-fold increase in dose between the 100 mg dose (Cohort 2) and the 400 mg dose (Cohort 4) on Day 1. Mean C_(max) values increased ˜3.0-fold over the 4-fold increase in dose between the 100 mg dose (Part 2, Cohort 2) and the 400 mg dose (Part 2, Cohort 4) on Day 14.

Systemic exposure to KAR5585, as measured by mean AUC₀₋₁₂ and AUC₀₋₂₄, appeared to increase in a dose-proportional manner between the 100 and 400 mg dose levels, with a 5.1-fold and 3.6-fold increase in mean AUC₀₋₁₂ and mean AUC₀₋₂₄ estimates, respectively, for a 4-fold increase in dose between the 100 mg dose (Part 2, Cohort 2) and the 400 mg dose (Part 2, Cohort 4) on Day 1. Mean AUC₀₋₁₂ and mean AUC₀₋₂₄ estimates, increased 3.1-fold and 3.63-fold respectively, for a 4-fold increase in dose between the 100 mg dose (Part 2, Cohort 2) and the 400 mg dose (Part 2, Cohort 4) on Day 14.

The mean apparent elimination half-life of KAR5585 increased with increasing dose levels studied, and the mean values were 2.73, 4.08 and 6.45 hrs on Day 14 after oral administration of repeat dose twice-daily administration of 100, 200 and 400 mg of KAR5585 (Part 2, Cohort 2-4,fed state), respectively. The half-life of KAR5585 was 2.49 hrs on Day 14 after oral administration of repeat dose twice-daily administration of 100 mg in the fasted state, and was comparable to the half-life in the fed state (2.73 hrs) for the 100 mg dose.

Peak plasma KAR5417 concentrations, as measured by mean C_(max), appeared to increase in a greater that dose proportional manner from 100-400 mg (Part 2, Cohort 2-4, fed state). Mean C_(max) values increased 6.9-fold over the 4-fold increase in dose between the 100 mg dose (Cohort 2) and the 400 mg dose (Cohort 4) on Day 1. Day 14 mean C_(max) values increased in an approximately dose proportional manner with an ˜4.5-fold increase in C_(max) over the 4-fold increase in dose between the 100 mg dose (Part 2, Cohort 2) and the 400 mg dose (Part 2, Cohort 4).

Mean extent of systemic exposure to KAR5417, as measured by mean AUC₀₋₁₂ and AUC₀₋₂₄, appeared to increase in a greater than dose-proportional manner between the 100 and 400 mg dose levels, with a 7.0-fold and 6.45-fold increase in mean AUC₀₋₁₂ and mean AUC₀₋₂₄ estimates, respectively, for a 4-fold increase in dose between the 100 mg dose (Part 2, Cohort 2) and the 400 mg dose (Part 2, Cohort 4) on Day 1. Day 14 mean AUC₀₋₁₂ and mean AUC₀₋₂₄ estimates, increased 4.6-fold and 4.45-fold respectively, for a 4-fold increase in dose between the 100 mg dose (Part 1, Cohort 2) and the 400 mg dose (Part 2, Cohort 4).

Overall, the mean extent of systemic exposure to KAR5417, as measured by mean AUC₀₋₁₂ and AUC₀₋₂₄, appeared to be comparable under fasted conditions (Part 2, Cohort 1; Day 1 AUC₀₋₁₂=741 hr*ng/mL; Day 14 AUC₀₋₂₄=1650 hr*ng/mL,) relative to fed conditions (Part 2, Cohort 2; Day 1 AUC₀₋₁₂=470 hr*ng/mL; Day 14 AUC₀₋₂₄=1220 hr*ng/mL,). This observation is in contrast to the food effect comparison in Part 1, Cohort 3A and 3B (400 mg KAR5585). In Part 1, a high fat, high calorie meal resulted in AUC_(0-inf) of KAR5417 that was approximately 1.8-fold higher following drug administration of KAR5585 400 mg under fed conditions (AUC_(0-inf)=6710 ng·hr/mL) relative to fasting conditions (AUC_(0-inf)=3650 ng·hr/mL).

Steady-state plasma levels of KAR5417 were achieved by Day 7 as assessed by comparison of Day 7 and Day 14 AUC₀₋₁₂ and C_(12 hr) values following twice daily oral administration of KAR5585 for 14-days. Accumulation ratios for KAR5417 compared R_(Cmax) and R_(AUC0-12) for Day 7/Day 1 and Day 14/Day 1, and verified that steady-state was achieved by Day 7. The R_(AUC0-12) values for KAR5417 ranged from 2.19-2.57, 1.09-1.34 and 1.65-2.38 for the 100, 200 and 400 mg doses of KAR5585, respectively, suggesting that the PK of KAR5417 was independent of time, and steady-state was achieved on Day 7 following repeated twice-daily administration for 14-days.

Pharmacokinetics Conclusions:

KAR5585 is a prodrug for the active TPH1 inhibitor KAR5417. Following KAR5585 administration, KAR5585 was rapidly absorbed and converted to KAR5417.

Peak plasma KAR5417 concentrations, as measured by mean C_(max), appeared to increase in a greater that dose proportional manner from 100-400 mg (Part 2, Cohort 2-4, fed state). Mean C_(max) values increased 6.9-fold over the 4-fold increase in dose between the 100 mg dose (Cohort 2) and the 400 mg dose (Cohort 4) on Day 1. On Day 14, mean C_(max) values increased in an approximately dose proportional manner with an ˜4.5-fold increase in C_(max) over the 4-fold increase in dose between the 100 mg dose (Part 2, Cohort 2) and the 400 mg dose (Part 2, Cohort 4). For doses of 100, 200 and 400 mg KAR5585, Part 2, Cohorts 2-4 (fed state), the median time to peak plasma concentrations of KAR5417 ranged from 2 to 6 hours, 3 to 8 hours, and 3 to 6 hrs.

Mean extent of systemic exposure to KAR5417, as measured by mean AUC₀₋₁₂ and AUC₀₋₂₄, appeared to increase in a greater than dose-proportional manner between the 100 and 400 mg dose levels, with a 7.0-fold and 6.45-fold increase in mean AUC₀₋₁₂ and mean AUC₀₋₂₄ estimates, respectively, for a 4-fold increase in dose between the 100 mg dose (Part 2, Cohort 2) and the 400 mg dose (Part 2, Cohort 4) on Day 1. Day 14, mean AUC₀₋₁₂ and mean AUC₀₋₂₄ estimates, increased 4.6-fold and 4.45-fold respectively, for a 4-fold increase in dose between the 100 mg dose (Part 1, Cohort 2) and the 400 mg dose (Part 2, Cohort 4).

Overall, the mean extent of systemic exposure to KAR5417, as measured by mean AUC₀₋₁₂ and AUC₀₋₂₄, appeared comparable under fasted conditions (Part 2, Cohort 1; Day 1 AUC₀₋₁₂=741 hr*ng/mL; Day 14 AUC₀₋₂₄=1650 hr*ng/mL,) relative to fed conditions (Part 2, Cohort 2; Day 1 AUC₀₋₁₂=470 hr*ng/mL; Day 14 AUC₀₋₂₄=1220 hr*ng/mL,). This observation is in contrast to the food effect comparison in Part 1, Cohort 3A and 3B (400 mg KAR5585). In Part 1, a high fat, high calorie meal resulted in AUC_(0-inf) of KAR5417 that was approximately 2-fold higher following drug administration of KAR5585 400 mg under fed conditions (AUC_(0-inf)=6710 ng·hr/mL) relative to fasting conditions (AUC_(0-inf)=3650 ng·hr/mL).

The mean apparent elimination half-life of KAR5417 was 19.6, 25.0 and 30.6 hrs on Day 14 after oral administration of repeat dose twice-daily administration of 100, 200 and 400 mg of KAR5585 (Part 2, Cohort 2-4, fed state). The elimination half —life (30.6 hrs) of KAR5417 on repeat dose administration of 400 mg doses of KAR5585, was similar to the elimination half-life of KAR5417 (20.5 hrs) following single-dose administration of 400 mg KAR5585 (Part 1 Cohort 3B).

KAR5417 plasma levels achieved steady-state exposure by Day 7 as assessed by comparison of Day 7 and Day 14 AUC₀₋₁₂ and C_(12 hr) values following twice daily administration of KAR5585 for 14-days. Accumulation ratios for KAR5417 compared R_(Cmax) and R_(AUC0-12) for Day 7/Day 1 and Day 14/Day 1. The R_(AUC0-12) values for KAR5417 ranged from 2.19-2.57, 1.09-1.34 and 1.65-2.38 for the 100, 200 and 400 mg doses of KAR5585 suggesting that the PK of KAR5417 was independent of time and steady-state was achieved on Day 7 following repeated, twice-daily administration for 14-days.

Biomarker Evaluation:

Biomarker evaluations were carried out in subjects dosed with KAR5585 or placebo.

The biomarker portion of the study was conducted in Part 2 MAD using the Biomarker Population. The biomarkers evaluated were serum 5-HT and plasma 5-HIAA, as well as urine 5-HIAA/24 hours (as measured, adjusted, estimated, and per gram creatinine). Urinary 5-HIAA was determined by a validated (21 C.F.R. 58, GLP-compliant) method using liquid chromatographic separation and tandem mass spectrometry detection.

The Biomarker Population consisted of all subjects with evaluable baseline (Day 1 predose) and at least one postdose (Day 7 or Day 14) biomarker measurements.

A total of 48 subjects received study drug according to the protocol (Table 22). Two plasma 5 HIAA values were excluded from analyses because Day 7 assessments were lacking. These were values for Subject 001-M036 in the KAR5585 200 mg fed Cohort and Subject 001-M001 in the KAR5585 100 mg fasting Cohort. Tables and figures missing a Day 7 value for one or both these subjects are marked with an “s”. Values excluded were identified based on statistical criteria and clinical sensibility.

Baseline Characteristics:

Baseline characteristics are presented in Table 23. Baseline levels of the biomarkers serum 5-HT, plasma-5HIAA, the 5-HIAA measured in urine during 24 hours, without adjustment for daily variations in creatinine excretion (measured urine 5-HIAA), the 5-HIAA measured in urine during 24 hours, adjusted for the mean 24-hour creatinine excretion on Days 1, 7, and 14 (adjusted urine 5-HIAA), estimated urine 5-HIAA, and urine 5-HIAA were similar among the 5 treatment groups (placebo, KAR5585 100 mg fasting, KAR5585 100 mg fed, KAR5585 200 mg fed, and KAR5585 400 mg fed).

Analysis of Biomarkers: Biomarker Results and Tabulations of Individual Subject Data:

Biomarker data were analyzed for each KAR5585 dose separately, for all KAR5585 doses pooled, and for all placebo subjects pooled.

Biomarker analyses were performed for:

-   -   Serum 5-HT     -   Plasma 5-HIAA     -   Urine 5-HIAA/24 hours (as measured, adjusted, estimated, and per         gram creatinine)

Analyses of biomarker changes and biomarker changes with respect to PK parameters were performed. Linear mixed-effect modeling for combined Day 7 and Day 14 biomarker measurements versus corresponding PK parameters to account for the repeated measures for the same subject had been planned but it was not performed because the relationships between Day 7 and Day 14 versus PK parameters were not consistent with each other.

Primary Biomarker Endpoint: Change in Plasma 5 HydroxyindoleaceticAcid from Day 1 to Day 14:

Dosing with KAR5585 reduced plasma 5-HIAA concentration. Mean percent change in plasma 5-HIAA was −56.67 from Day 1 to Day 7 and −53.33 from Day 1 to Day 14 in subjects randomized to KAR5575 400 mg under fed conditions. Mean percent change at Day 7 was +19.79 and at Day 14 was +20.12 in subjects randomized to placebo. Mean difference of both absolute and percent changes from Day 1 to Day 14 in plasma 5-HIAA concentration was statistically significant in favor of KAR5585 compared to placebo in each dose group and for all doses combined at Day 14 (Table 17).

TABLE 17 (Differences of Changes from Day 1 Predose between KAR5585 and Placebo of Plasma 5-HIAA (ng/ml) by Cohort-Part 2 MAD Biomarker Population) Mean Difference of Changes from Day 1 between KAR5585 and Placebo Absolute Change from Day 1 % Change from Day 1 Estimate Estimate Dose Level/Time (95% CI) P Value (95% CI) P Value 100 mg Predose Day 7 0.33 0.9531 23.67 0.6330 Fasting (−10.74, 11.41) (−73.11, 120.45) Predose Day 14 −5.92 0.0015 −48.83 <.0001 (−9.42, −2.41) (−67.43, −30.22) 100 mg Predose Day 7 −4.56 0.4226 −48.56 0.3284 Fed (−15.63, 6.52) (−145.34, 48.21) Predose Day 14 −4.50 0.0175 −52.70 <.0001 (−8.13, −0.87) (−71.95, −33.45) 200 mg Predose Day 7 1.22 0.8293 12.06 0.8077 Fed (−9.85, 12.30) (−84.72, 108.83) Predose Day 14 −5.25 0.0059 −52.46 <.0001 (−8.88, −1.62) (−71.71, −33.20) 400 mg Predose Day 7 −11.44 0.0463 −76.47 0.1255 Fed (−22.52, −0.37) (−173.24, 20.31) Predose Day 14 −10.69 <.0001 −73.45 <.0001 (−14.20, −7.19) (−92.05, −54.85) All Predose Day 7 −3.61 0.4005 −22.33 0.5515 Doses (−11.98, 4.76) (−95.48, 50.83) Predose Day 14 −6.69 <.0001 −57.11 <.0001 (−9.36, −4.02) (−71.27, −42.95) Abbreviations: 5-HIAA, 5-hydroxyindoleacetic acid; CI, confidence interval; MAD, multiple ascending dose.

Biomarker: Measured Urine 5-Hydroxyindoleacetic Acid:

Urine 5 HIAA/24 hour was analyzed in 4 different manners: as measured in the 24-hour collection and (in order to correct for any collection errors) adjusted for creatinine excretion, estimated by the expected creatinine excretion for the subject (as defined in and expressed per gram creatinine. Results were similar for each of the 4 manners and are presented in this report for measured urine. Tables and figures for adjusted, estimated, and per-gram creatinine results are presented in Section 14.

Dosing with KAR5585 reduced measured urine 5-HIAA concentration. Mean percent change in measured urine 5-HIAA was −39.97 from Day 1 to Day 7 and −50.85 from Day 1 to Day 14 in subjects randomized to KAR5575 400 mg under fed conditions. Mean percent change at Day 7 was +9.44 and at Day 14 was +3.97 in subjects randomized to placebo.

Difference of absolute and relative changes from Day 1 in measured urine 5-HIAA on Days 7 and 14 were statistically significant in favor of KAR5585 in all dose groups except one: the relative change on Day 14 for the KAR5585 100 mg fasting dose group (Table 18).

TABLE 18 (Differences of Changes from Day 1 Predose between KAR5585 and Placebo of Measured^(a) Urine 5-Hydroxyindoleacetic Acid (mg/24 hours), by Cohort (Biomarker Population)) Mean Difference of Changes from Day 1 between KAR5585 and Placebo Absolute Change from Day 1 % Change from Day 1 Estimate Estimate Dose Level/Time (95% CI) P Value (95% CI) P Value 100 mg Predose Day 7 −0.92 .0208 −31.97 .0005 Fasting (−1.68, −0.16) (−49.29, −14.64) Predose Day 14 −0.91 .0007 −17.65 .1113 (−1.41, −0.40) (−39.12, 3.82) 100 mg Predose Day 7 −2.09 <.0001 −50.34 <.0001 Fed (−2.86, −1.33) (−67.66, −33.01) Predose Day 14 −1.65 <.0001 −36.03 .0022 (−2.18, −1.13) (−58.26, −13.81) 200 mg Predose Day 7 −0.92 .0248 −26.95 .0043 Fed (−1.71, −0.13) (−44.89, −9.02) Predose Day 14 −1.58 <.0001 −37.11 .0016 (−2.10, −1.06) (−59.33, −14.89) 400 mg Predose Day 7 −1.68 <.0001 −49.41 <.0001 Fed (−2.44, −0.92) (−66.73, −32.08) Predose Day 14 −2.04 <.0001 −54.82 <.0001 (−2.55, −1.54) (−76.29, −33.35) All Predose Day 7 −1.42 <.0001 −40.03 <.0001 Doses (−2.00, −0.84) (−53.17, −26.89) Predose Day 14 −1.54 <.0001 −36.39 <.0001 (−1.93, −1.16) (−52.74, −20.05) ^(a)Measured is as measured in 24-hour 5HIAA excretion without adjustment for variations in daily creatinine excretion. Abbreviations: 5-HIAA, 5-hydroxyindoleacetic acid, CI, confidence interval.

Both absolute and percent changes from Day 1 in creatinine-normalized urine 5-HIAA on Days 7 and 14 were statistically significant in favor of KAR5585 versus placebo in all dose groups.

Serum 5-Hydroxytryptamine:

Mean difference of both absolute and relative changes from Day 1 to Day 14 in serum 5-HT concentration was statistically significant in favor of KAR5585 compared to placebo for the 400 mg fed and all KAR5585 doses pooled (Table 19).

Mean difference of percent changes from Day 1 in serum 5-HT concentration was statistically significant compared to placebo on Day 7 in favor of the 200 mg fed dose group.

TABLE 19 Differences of Changes from Day 1 Predose between KAR5585 and Placebo of (Serum 5-HT (ng/ml) by Cohort-Part 2 MAD Biomarker Population) Mean Difference of Changes from Day 1 between KAR5585 and Placebo Absolute Change from Day 1 % Change from Day 1 Estimate Estimate Dose Level/Time (95% CI) P Value (95% CI) P Value 100 mg Predose Day 7 −12.97 .4642 −10.97 .2567 Fasting (−47.53, 21.59) (−29.80, 7.85) Predose Day 14 −17.39 .3285 −15.92 .1598 (−52.04, 17.26) (−37.90, 6.06) 100 mg Predose Day 7 −15.75 .3745 −11.02 .2549 Fed (−50.31, 18.81) (−29.84, 7.81) Predose Day 14 5.08 .7820 −0.47 .9678 (−30.79, 40.95) (−23.22, 22.28) 200 mg Predose Day 7 −28.53 .1097 −21.64 .0270 Fed (−63.09, 6.03) (−40.47, −2.82) Predose Day 14 −5.79 .7525 −11.43 .3279 (−41.66, 30.08) (−34.18, 11.32) 400 mg Predose Day 7 16.47 .3531 2.09 .8282 Fed (−18.09, 51.03) (−16.73, 20.91) Predose Day 14 −84.50 <.0001 −50.02 <.0001 (−119.15, −49.85) (−72.00, −28.04) All Predose Day 7 −10.19 .4467 −10.39 .1566 Doses (−36.32, 15.93) (−24.61, 3.84) Predose Day 14 −27.14 .0475 −20.26 .0203 (−53.52, −0.75) (−36.99, −3.52)

Analysis of Biomarkers in Relation to Pharmacokinetic Parameters:

All subjects were dosed with KAR5585 or placebo. Following dosing, KAR5585 was rapidly absorbed and the active moiety KAR5417 appeared rapidly in plasma.

Only those subjects with all biomarker results and results for the PK parameters AUC₀₋₂₄, C_(min), concentration, obtained 12 hr after an administered dose (C₁₂ hr), and AUC₀₋₁₂ were included in the PK-Biomarker analyses. The PK parameters AUC₀₋₂₄, C_(min), C12 _(hr), and AUC₀₋₁₂ were adjusted from those stated in the protocol (AUC₀₋₂₄, C_(min), and C_(max)) to account for the double daily dosing.

All subjects in Part 2 (MAD) were eligible for biomarker analyses. Only the subset of the Biomarker Population randomized to active drug were included in PK-Biomarker analyses.

Pharmacokinetic Parameters and Plasma 5-Hydroxyindoleacetic Acid, the Primary Biomarker Endpoint:

On Day 14, the absolute and relative changes from baseline in plasma 5-HIAA were significantly correlated with KAR5417 AUC₀₋₂₄, C_(max), C₁₂, and AUC₀₋₁₂ (P≤0.0010).

Greater values for the PK parameters were associated with greater decreases in plasma 5-HIAA concentrations, as illustrated in FIG. 23 , which shows the relationship between relative changes from baseline in plasma 5-HIAA and KAR5417 AUC₀₋₂₄.

FIG. 23 discloses absolute and relative changes at day 14 of Plasma 5-HIAA from Day 1 Predose vs. AUC_([0-24]) in subjects who received KAR5585—Part 2 MAD Biomarker Population.

While there were no significant correlations on Day 7 between absolute or relative change from baseline in serum 5-HT concentration and AUC₀₋₂₄, C_(max), C₁₂, or AUC₀₋₁₂ for KAR5417, both absolute and relative changes on Day 14 were correlated (P<0.0010).

While there were no significant correlations between absolute or relative change from baseline in measured urine 5-HIAA and KAR5417 AUC₀₋₂₄, C_(max), C₁₂, or AUC₀₋₁₂ on Day 7, both absolute and relative changes were significantly correlated for Day 14 (P≤0.0067). Greater values for the PK parameters were associated with greater decreases in measured urine 5-HIAA concentrations, which shows the relationship between relative change in measured urine 5-HIAA (mg/24 hours) from Day 1 to Day 14 and KAR5417 AUC_(0-24.)

The relationships between adjusted and estimated urine 5-HIAA and KAR5417 PK parameters were similar to those for measured urine 5-HIAA: though there were no significant correlations on Day 7 between absolute or relative change from baseline in urine 5-HIAA and KAR5417 PK parameters, the correlations were significant on Day 14 for both absolute and relative changes in urine 5-HIAA and KAR5417 PK parameters (adjusted urine 5-HIAA, P≤0.0007; estimated urine 5-HIAA, P≤0.0110).

Discussion of Biomarker Results:

There was a strong association between plasma 5-HIAA (pre-established as the primary biomarker endpoint) and both KAR5585 dose and the duration of dosing. The mean reduction from Day 1 to Day 14 in plasma 5-HIAA was greater for the highest dose (

53.33%, 400 mg fed) than for the lowest dose (−28.71%, 100 mg fasting; −32.58%, 100 mg fed). The mean percent difference of change from Day 1 between KAR5585 and placebo was significant (P<0.0001) for each dose group and all doses pooled at Day 14 but not significant at Day 7 for any dose group.

There was also a strong association between urine 5-HIAA/24 hours and KAR5585 dose. At the highest KAR5585 dose, 400 mg, mean percent change in measured urine 5-HIAA was 1150.85 from Day 1 to Day 14, whereas mean percent change in subjects randomized to placebo was +3.97. Mean differences of absolute and relative changes from Day 1 to Day 14 were statistically significant in favor of KAR5585 in all dose groups except one (the relative change on Day 14 for the KAR5585 100 mg fasting dose group). Results were comparable in urine 5-HIAA/24 hours as adjusted, estimated, and per gram creatinine.

Serum 5-HT did not reveal the strong association between the biomarker of serotonin and KAR5585 dose or duration of dosing. The differences of changes from Day 1 between KAR5585 and placebo were statistically significant for the highest dose group (400 mg fed) and for all KAR5585 doses pooled.

A strong relationship was seen between KAR5417 exposure and 5-HIAA reductions. A strong relationship between both plasma and urine 5-HIAA is supported by the observation that higher exposure to KAR5417, as measured by the PK parameters AUC₀₋₂₄, C_(max), C₁₂, and AUC₀₋₁₂, was associated with greater reduction in 5-HIAA.

Biomarker Conclusions:

Dose- and time-dependent reductions were observed in 5-HIAA (a PD marker of 5-HT synthesis) in both plasma and urine.

At the highest KAR5585 dose, 400 mg, mean percent change in plasma 5-HIAA concentration was −53.33 from Day 1 to Day 14, whereas mean percent change in subjects randomized to placebo was +20.12.

At the highest KAR5585 dose, 400 mg, mean percent change in measured urine 5-HIAA was −50.85 from Day 1 to Day 14, whereas mean percent change in subjects randomized to placebo was +3.97. Results were comparable in urine 5-HIAA/24 hours as adjusted, estimated, and per gram creatinine.

A strong relationship was seen between KAR5417 exposure and 5-HIAA reductions.

OVERALL CONCLUSIONS

The study was divided into 2 parts: SAD (Part 1) and MAD (Part 2). Part 1 (SAD) was further divided into Period 1 (fasting administration) and Period 2 (fed administration, food effect). The KAR5585 doses administered were the following:

-   -   In Part 1 (SAD) Period 1 (fasting): 100 mg, 200 mg, 400 mg, 700         mg, 1200 mg, or 2000 mg in Cohorts 1 through 6, respectively     -   In Part 1 (SAD) Period 2 (fed, high-fat food effect): 400 mg in         Cohort 3 only     -   In Part 2 (MAD): 100 mg (fasting), 100 mg (fed), 200 mg (fed),         and 400 mg (fed) or matching placebo administered BID         (approximately every 12 hours) for 27 doses in Cohorts 1 through         4, respectively.

Pharmacokinetics: Following administration of KAR5585 (prodrug), KAR5417 (active TPH1 inhibitor) appeared rapidly in plasma, an observation consistent with rapid absorption of KAR5585 and efficient conversion to KAR5417.

Administration of the KAR5585 under fed conditions in Part 1, Period 2, increased the extent and peak of exposure of both KAR5585 and KAR5417. This was considered to be a clinically-relevant change in exposure. In Part 2, the mean extent of systemic exposure to KAR5417, as measured by mean AUC₀₋₁₂ and AUC₀₋₂₄, appeared comparable under fasted conditions. This observation is in contrast to the food-effect comparison in Part 1.

Biomarkers: Dose- and time-dependent reductions were observed in 5-HIAA (a PD marker of 5-HT synthesis) in both plasma and urine.

At the highest KAR5585 dose, 400 mg, mean percent change in plasma 5-HIAA concentration was −53.33 from Day 1 to Day 14, whereas mean percent change in subjects randomized to placebo was +20.12.

At the highest KAR5585 dose, 400 mg, mean percent change in measured urine 5-HIAA was −50.85 from Day 1 to Day 14, whereas mean percent change in subjects randomized to placebo was +3.97. Results were comparable in urine 5-HIAA/24 hours as adjusted, estimated, and per gram creatinine.

A strong relationship was seen between KAR5417 exposure and 5-HIAA reductions.

Following administration of KAR5585 (prodrug), KAR5417 (active TPH1 inhibitor) appeared rapidly in plasma, an observation consistent with rapid absorption of KAR5585 and efficient conversion to KAR5417. A strong relationship was seen between KAR5417 exposure and 5-HIAA reductions. Neither the prodrug (KAR5585) nor the active drug (KAR5417) showed any tendency to increase QTcF in a dose-dependent manner.

It should be understood that the foregoing description is only illustrative of the present disclosure. Various alternatives and modifications can be devised by those skilled in the art without departing from the present disclosure. Accordingly, the present disclosure is intended to embrace all such alternatives, modifications and variances which fall within the scope of the appended claims. 

What is claimed is:
 1. A daily dosage regimen for treating pulmonary arterial hypertension, comprising two discrete dosage forms, wherein the dosage forms each include an amount of about 600 mg to about 800 mg of (S)-ethyl 8-(2-amino-6-((R)-1-(5-chloro-[1,1′-biphenyl]-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylate.
 2. The daily dosage regimen of claim 1, wherein the dosage forms each include RVT-1201, wherein the (S)-ethyl 8-(2-amino-6-((R)-1-(5-chloro-[1,1′-biphenyl]-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylate is substantially as depicted in FIG. 1 .
 3. The daily dosage regimen of claim 1, wherein the dosage forms are oral dosage forms.
 4. The daily dosage regimen of claim 1, wherein each of the dosage forms further include an amount of a pharmaceutically acceptable excipient.
 5. A daily dosage regimen for treating pulmonary arterial hypertension, comprising an amount of 1200 mg to 1600 mg of (S)-ethyl 8-(2-amino-6-((R)-1-(5-chloro-[1,1′-biphenyl]-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylate.
 6. The daily dosage regimen of claim 5, wherein the (S)-ethyl 8-(2-amino-6-((R)-1-(5-chloro-[1,1′-biphenyl]-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylate is substantially as depicted in claim
 1. 7. The daily dosage regimen of claim 6, wherein the amount further includes a pharmaceutically acceptable excipient.
 8. A method for treating pulmonary arterial hypertension, comprising administering to a human patient in need thereof an amount of 1200 mg to 1600 mg of (S)-ethyl 8-(2-amino-6-((R)-1-(5-chloro-[1,1′-biphenyl]-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylate per day.
 9. The method of claim 8, wherein the (S)-ethyl 8-(2-amino-6-((R)-1-(5-chloro-[1,1′-biphenyl]-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylate is substantially as depicted in FIG. 1 .
 10. The method of claim 8, wherein the amount is administered orally.
 11. The method of claim 8, wherein the amount is administered at 600 mg to 800 mg BID.
 12. A method for reducing the level of serotonin biosynthesis by at least 50% within 14 days after commencement of treatment, comprising administering to a human patient in need thereof an amount of about 800 mg to about 1600 mg of (S)-ethyl 8-(2-amino-6-((R)-1-(5-chloro-[1,1′-biphenyl]-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylate per day.
 13. The method of claim 12, wherein the (S)-ethyl 8-(2-amino-6-((R)-1-(5-chloro-[1,1′-biphenyl]-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylate is substantially as depicted in FIG. 1 .
 14. The method of claim 12, wherein the amount is administered orally.
 15. The method of claim 12, wherein the amount is administered is about 400 mg to about 800 mg BID.
 16. The method of claim 12, wherein the amount is administered at dosages selected from the group consisting of about 400 mg BID, 600 mg BID, and 800 mg BID.
 17. A method for achieving an AUC_(0-tau) of ≥2530 ng.hr/mL of (S)-8-(2-amino-6-((R)-1-(5-chloro-[1,1′-biphenyl]-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylic acid within 14 days after administration, comprising administering daily to a human patient an effective amount of (S)-ethyl 8-(2-amino-6-((R)-1-(5-chloro-[1,1′-biphenyl]-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylate.
 18. The method of claim 17, wherein the amount administered is about 800 mg to about 1600 mg per day.
 19. The method of claim 18, wherein the amount is administered is about 400 mg to about 800 mg BID.
 19. A method of achieving a >50% reduction in urinary 5-HIAA within 14 days after administration, comprising administering daily to a human patient an effective amount of (S)-ethyl 8-(2-amino-6-((R)-1-(5-chloro-[1,1′-biphenyl]-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylate.
 20. The method of claim 19, wherein the amount administered is about 800 mg to about 1600 mg per day.
 21. The method of claim 20, wherein the amount is administered is about 400 mg to about 800 mg BID.
 22. A method for treating pulmonary arterial hypertension, comprising administering daily to a human patient in need thereof an amount of (S)-ethyl 8-(2-amino-6-((R)-1-(5-chloro-[1,1′-biphenyl]-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylate sufficient to achieve an AUC_(0-tau) of ≥2530 ng.hr/mL of (S)-8-(2-amino-6-((R)-1-(5-chloro-[1,1′-biphenyl]-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylic acid within 14 days after commencement of administration.
 23. A method for treating pulmonary arterial hypertension, comprising administering daily to a human patient in need thereof an amount of (S)-ethyl 8-(2-amino-6-((R)-1-(5-chloro-[1,1′-biphenyl]-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylate effective to achieve a >50% reduction in urinary 5-HIAA within 14 days after commencement of administration. 